Genetically engineered BPI variant proteins

ABSTRACT

The present invention provides a composition comprising a BPI Protein and an anionic compound which composition exhibits (1) no bactericidal activity and (2) endotoxin neutralizing activity. Also, this invention provides methods for using BPI Proteins.

This application is a 35 U.S. C 371 of PCT/US91/05758 filed Aug. 13,1991 and a continuation-in-part of U.S. Ser. No. 567,016, filed Aug. 13,1990, now abandoned and U.S. Ser. No. 681,551, filed Apr. 5, 1991, nowabandoned. U.S. Pat. No. 5,171,739, the contents of all of which arehereby incorporated by reference.

BACKGROUND OF THE INVENTION

Gram negative infections are a major cause of morbidity and mortalityespecially in hospitalized and immunocompromised patients. Duma, R. J.,Am. J. of Med., 78 (Suppl. 6A): 154-164 (1985); and Kreger B. E., D. E.Craven and W. R. McCabe, Am. J. Med., 68: 344-355 (1980)!. Althoughavailable antibiotics are generally effective in containing theinfection, they do nothing to neutralize the pathophysiological effectsassociated with lipopolysaccharide (LPS).

LPS is a major component of the outer membrane of gram negative bacteriaand is released when the organisms are lysed. Shenep, J. L. and K. A.Morgan, J. Infect. Dis., 150 (3): 380-388 (1984)!

LPS released during antibiotic therapy is a potent stimulator of theinflammatory response. Many detrimental effects of LPS in vivo resultfrom soluble mediators released by inflammatory cells. Morrison D. C.and R. J. Ulevich, Am. J. Pathol., 93 (2): 527-617 (1978)! LPS inducesthe release of mediators by host inflammatory cells which may ultimatelyresult in disseminated intravascular coagulation (DIC), adultrespiratory distress syndrome (ARDS), cardiac dysfunction, organfailure, liver failure (hepatobiliary dysfunction), brain failure (CNSdysfunction), renal failure, multi-organ failure and shock.

Soluble LPS causes decreased neutrophil chemotaxis, increasedadhesiveness, elevated hexose monophosphate shunt activity and O₂radical production, upregulation of surface receptors for complement,and release of granule proteins into the surrounding medium. Morrisonand Ulevich (1978)!

Endotoxemia is a condition associated with the presence of endotoxins,i.e. heat stable bacterial toxins, in the blood. Endotoxins elicit aninflammatory response that is beneficial in fighting the infection butcan be damaging to the host if uncontrolled. Endotoxemia inducesproduction of endotoxin binding proteins from the liver and causesrelease of microbicidal proteins from leukocytes. Our studies show thatone of these leukocytes proteins, i.e. BPI, previously known only forits bactericidal activity in vitro. inhibits the ability of endotoxin tostimulate neutrophils and monocytes Ln vitro and reduces death due toendotoxin or bacterial challenge when given in vivo. Further, BPI hasbeen shown to possess antibiotic functions but not cytotoxin functionsagainst the host cell.

Monocytes and neutrophilic granulocytes play a key role in host defenseagainst bacterial infections and also participate in the pathology ofendotoxemia. These cells ingest and kill microorganisms intracellularlyand also respond to endotoxin in vivo and in vitro by releasing solubleproteins with microbicidal, proteolytic, opsonic, pyrogenic, complementactivating and tissue damaging effects.

Tumor necrosis factor (TNF), a cytokine released by endotoxin stimulatedmonocytes mimics some of the toxic effects of endotoxin in vivo.Injecting animals with TNF causes fever, shock and alterations inglucose metabolism. TNF is also a potent stimulator of neutrophils.Other cytokines such as IL-1, IL-6, and IL-8 also mediate some of thepathophysiologic effects of LPS.

Despite improvements in antibiotic therapy, morbidity and mortalityassociated with endotoxemia remains high. Antibiotics alone are noteffective in neutralizing the toxic effects of LPS. Therefore, the needarises for a therapy with direct endotoxin neutralizing activity.Current methods for treatment of endotoxemia use antibiotics andsupportive care. Most available adjunct therapies treat symptoms ofendotoxic shock such as low blood pressure and fever but do notinactivate endotoxin. Other therapies inhibit inflammatory hostresponses to LPS. As indicated below, present therapies have majorlimitations due to toxicity, immunogenicity, or irreproducible efficacybetween animal models and human trials.

Polymyxin B (PMB) is a basic polypeptide antibiotic which has been shownto bind to, and structurally disrupt, the most toxic and biologicallyactive component of endotoxin, Lipid A. PMB has been shown to inhibitendotoxin activation of neutrophil granule release in vitro and is apotential treatment for gram negative infections in humans. However,because of its systemic toxicity, this drug has limited use except as atopical agent.

Combination therapy using antibiotics and high doses ofmethylprednisolone sodium succinate (MPSS) has been shown to preventdeath in an experimental model of gram negative sepsis using dogs.Another study using MPSS with antibiotics in a multicenter, doubleblind, placebo-controlled, clinical study in 223 patients with clinicalsigns of systemic sepsis concluded that mortality was not significantlydifferent between the treatment and placebo groups. Further, theinvestigators found that resolution of secondary infection within 14days was significantly higher in the placebo group.

A relatively new approach to treatment of endotoxemia is passiveimmunization with endotoxin neutralizing antibodies. Hyperimmune humanimmunoglobulin against E. Coli J5 has been shown to reduce mortality inpatients with gram negative bacteremia and shock by 50%. Other groupshave shown promising results in animal models using mouse, chimeric, andhuman monoclonal antibodies. Although monoclonal antibodies haveadvantages over hyperimmune sera, e.g. more consistent drug potency anddecreased transmission of human pathogens, there are still many problemsassociated with administering immunoglobulin to neutralize LPS. Hostresponses to the immunoglobulins themselves can result inhypersensitivity. Tissue damage following complement activation anddeposition of immune complexes is another concern in the use oftherapies involving anti-endotoxin antibodies in septic patients.

BPI is a neutrophil granule protein first discovered in 1975 Weiss, J.,R. C. Franson, S. Becherdite, K. Schmeidler, and P. Elsbach J. Clin.Invest., 55:33 (1975)!. BPI was obtained in highly purified form fromhuman neutrophils in 1978 and was shown to increase membranepermeability and have bactericidal activity against Gram negativebacteria when assayed in phosphate buffered saline in vitro Weiss, J.,et al. J. Biol. Chem,253(8): 2664-2672 (1978)!. Weiss et al. J. Biol.Chem. 254(21): 110010-11014 (1979)!, further showed that BPI increasedphospholipase A2 activity suggesting a proinflammatory activity for BPIin addition to its in vitro bactericidal activity.

Rabbit BPI was purified in 1979 Elsbach et al. J. Biol. Chem 254(21):11000-11009! and shown to have identical bactericidal and permeabilityincreasing properties as BPI from humans providing a further source ofmaterial for study. Both BPI from rabbit and human were shown to beeffective against a variety of Gram negative bacteria in vitro,including K1-encapsulated E. coli Weiss et al. Infection and Immunity38(3): 1149-1153, (1982)!.

A role for lipopolysaccharide in the in vitro bactericidal action of BPIwas proposed in 1984 by Weiss et al. J. Immunol. 132(6): 3109-3115,(1984)!. These investigators demonstrated that BPI bound to the outermembrane of gram-negative bacteria, caused extracellular release of LPS,and selectively stimulated LPS biosynthesis. In 1984 a protein withsimilar properties was isolated from human neutrophils and designatedcationic antimicrobial protein 57 (CAP 57) Shafer, W. M., C. E. Martinand J. K. Spitznagel, Infect. Immun., 45:29 (1984)! This protein isidentical to BPI as determined by the N-terminal amino acid sequence,amino acid composition, molecular weight and source Spitznagel et al.,Blood 76:825-834, 1990!. Another group, Hovde and Gray, reported abactericidal glycoprotein with virtually identical properties to BPI in1986 Hovde and Gray, Infection and Immunity 54(1): 142-148 (1986)!.

In 1985 Ooi et al. reported that BPI retains its in vitro bactericidalactivity after cleavage with neutrophil proteases suggesting thatfragments of the molecule retain activity Ooi and Elsbach, ClinicalResearch 33(2) :567A (1985)!. All of the in vitro bactericidal andpermeability increasing activities of BPI were present in the N-terminal25 kD fragment of the protein Ooi, C.E., et al. J. Biol. Chem. 262:14891 (1987)!

Evidence that BPI binds to a structure associated with endotoxin on theouter membrane of bacteria is as follows: (1) increased sensitivity ofrough strains of E. coli relative to smooth strains to the permeabilityincreasing activities of BPI Weiss, J. et al. Infect. Immun. 51:594(1986)!; (2) the Prm A mutation which results in altered endotoxinstructure caused decreased binding of both polymyzin b. and BPI Farley,M. M. et al. Infect. Immun. 56:1536-1539 (1987) and Farley et al.Infect. Immun. 58:1589-1592 (1988)!; (3) polymyxin B (PMB) completedwith BPI for binding to S. typhimurium Farley 1988!; and (4) BPI sharedamino acid sequence homology and immunocrossreactivity to anotherendotoxin binding protein termed Lipopolysaccharide Binding Protein(LBP) Tobias et al., J. Biol. Chem. 263(27): 13479-13481 (1988)!.

LBP-LPS complexes bind to a cell surface receptor on monocytes (CD 14)which results in increased synthesis and release of the inflammatorycytokine tumor necrosis factor (TNF) Schumann et al. Science249:1429-1431!. Thus, LBP promotes the immunostimulatory activities ofLPS. BPI has exactly the opposite effect of LBP. BPI binds LPS andinhibits neutrophil and monocyte activation Marra et al., J. Immunol.144:662-666 (1990); Marra and Scott, WO90/09183, published 23 August1990; C. J. Fisher et al. Circulatory Shock 34: 120 (1991)!.

A cDNA encoding BPI was obtained and sequenced by Gray et al. Gray etal. Clin. Res. 36:620A (1988) and Gray et al. J. Biol. Chem. 264(16):9505-9506 (1989)!. They reported that BPI is a membrane protein whichcan be cleaved and released in soluble form as a 25 kDa fragment.

BPI binding to gram negative bacteria was reported originally to disruptLPS structure, alter microbial permeability to small hydrophobicmolecules and cause cell death (Weiss, et al., 1978). More recentlythese same authors have demonstrated that such effects occur only in theabsence of serum albumin. BPI has no bactericidal activity when added tobacteria cultured in the presence of serum albumin, thus suggesting thatBPI does not kill bacteria in vivo where albumin is ubiquitous Mannionet al. J. Clin. Invest. 85: 853-860 (1990) and Mannion et al J. clin.Invest. 86: 631-641)!. Thus it has been previously understood in the artthat the beneficial effects of BPI are limited to in vitro bactericidaleffects.

Here we show that BPI binds endotoxin in the presence of serum andplasma and, unlike other known endotoxin binding proteins such as LBP,BPI inhibits the immunostimulatory and toxic activities of endotoxinboth in vitro and in vivo respectively. Thus BPI has a novel anddistinct use in the therapeutic and prophylactic treatment ofendotoxin-related disorders including endotoxemia and endotoxic shock.

Further, BPI is described by Gray et al. J. Biol. Chem. 264 (16):9505-9509 (1989)! as a membrane protein which must be cleaved to the 25kDa fragment to be released from the neutrophil granule membrane insoluble form. The present invention provides for a method of producingfull length soluble BPI in active form. Further the present inventionseparates for the first time two molecular forms of the moleculeapparently unresolved by Gray et al. representing glycosylated andnonglycosylated forms of the molecule which appear to have differentserum half-life profiles in vivo and thus different therapeuticpotential. BPI from neutrophils is a mixture of the glycoslyated andnonglycosylated forms.

SUMMARY OF THE INVENTION

The present invention provides a composition comprising a BPI Proteinand an anionic compound which composition exhibits (1) no bactericidalactivity and (2) endotoxin neutralizing activity.

This invention also provides a biologically active variant of BPI which(1) specifically binds to endotoxin, (2) competes with BPI Protein forbinding to endotoxin, and (3) inhibits endotoxin-induced lethality.

The present invention further provides a method for producing andsecreting a recombinant BPI Protein from a cell. This method comprises(a) constructing a vector comprising DNA encoding BPI Protein; (b)transfecting the cell with the vector; and (c) culturing the cell sotransfected in culture medium under conditions such that recombinant BPIProtein is secreted.

Also, the present invention provides a method for producing arecombinant BPI Protein from a bacterial cell. This method comprises (a)constructing a vector without a signal sequence and comprising DNAencoding BPI Protein; (b) transfecting the bacterial cell with thevector; and (c) culturing the bacterial cell so transfected in culturemedium under conditions such that recombinant BPI Protein is produced.

The subject invention further provides a method for producing arecombinant BPI Protein from an insect cell. This method comprises (a)constructing a vector comprising DNA encoding BPI Protein; (b)transfecting the insect cell with the vector; and (c) culturing theinsect cell so transfected in culture medium under conditions such thatBPI Protein is produced.

Also, this invention provides a method for determining the amount ofendotoxin in a sample from a subject which comprises contacting thesample with a BPI Protein under conditions such that an endotoxin-BPIProtein complex is formed, detecting the amount of the complex so formedthereby determining the amount of endotoxin in the sample.

Additionally, the present invention provides a method for determiningthe amount of endotoxin in a sample containing bound and unboundendotoxin from a subject. This method comprises (a) treating the sampleso as to denature any endotoxin binding protein to which the endotoxinmay be bound thereby obtaining unbound endotoxin; (b) contacting thetreated sample with a BPI Protein under conditions such that the BPIProtein binds to unbound endotoxin of step (a) so that a endotoxin-BPIProtein complex is formed; (c) detecting the amount of the complex soformed thereby determining the amount of endotoxin in the sample.

This present invention also provides a method of detecting endotoxin ina sample which comprises contacting the sample with a BPI Protein suchthat the endotoxin binds to the BPI Protein and forms a complextherewith; and detecting such complex.

The present invention further provides a method for coating a surgicaltool with a BPI Protein so that the BPI Protein will complex withendotoxin which method comprises attaching BPI Protein onto a surface ofthe tool which surface is designed for contact with a biological sample.

Also, this invention provides a method for coating an implantable,invasive device with a BPI Protein so that it will form a complex withendotoxin which method comprises attaching BPI Protein onto a surface ofthe device which surface is designed for contact with a biologicalsample.

The present invention further provides a method for decontaminating afluid containing endotoxin prior to administration of the fluid into asubject which comprises contacting the fluid with BPI Protein prior toadministration, under conditions such that endotoxin forms a complexwith BPI Protein, thereby decontaminating the fluid. The fluid may beblood, plasma, blood serum, an isotonic solution, a pharmaceuticalagent, a cell culture reagent, or bone marrow.

This invention also provides a kit for detecting the presence of BPIProtein in a biological fluid sample which comprises (a) an assay buffercontaining polymyxin B which binds unbound endotoxin molecules; (b) afirst antibody attached to a surface, which antibody (1) binds to aportion of active BPI Protein and (2) does not compete with BPI Proteinfor an endotoxin binding domain; and (c) a second antibody labeled witha detectable moiety which antibody (1) does not compete with the firstantibody for BPI Protein binding and (2) specifically binds to BPIProtein at or near the endotoxin binding site, so that when thebiological fluid sample is contacted with the first and second antibodyin assay buffer, an active BPI Protein contained in the biological fluidsample is bound by the first and second antibody thus forming a firstantibody-BPI Protein-second antibody complex, detecting such complex,and thereby detecting BPI Protein in the biological fluid sample.

Also, this invention provides a kit for determining the amount of BPIProtein in a biological fluid sample which comprises (a) an assay buffercontaining polymyxin B which binds unbound endotoxin molecules; (b) afirst antibody attached to a surface, which antibody (1) binds to aportion of active BPI Protein and (2) does not compete with BPI Proteinfor an endotoxin binding domain; and (c) a second antibody labeled witha detectable moiety which antibody (1) does not compete with the firstantibody for BPI Protein binding and (2) specifically binds to BPIProtein at or near the endotoxin binding site, so that when thebiological fluid sample is contacted with the first and second antibodyin assay buffer, an active BPI Protein contained in the biological fluidsample is bound by the first and second antibody thus forming a firstantibody-active BPI Protein-second antibody complex, detecting suchcomplex, and determining the amount of active BPI Protein in thebiological fluid sample.

Additionally, this invention provides a method for preventingendotoxemia in a subject which comprises administering to the subject anamount of a BPI Protein effective to bind to endotoxin so as to preventendotoxemia in the subject.

The present invention provides a method for treating a subject sufferingfrom endotoxemia which comprises administering to the subject an amountof a BPI Protein effective to bind endotoxin so as to treat the subjectsuffering from endotoxemia.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Photographs of transformed plates of JM109(DE3) with the T7promoter/BPI Protein plasmid constructs. Photographs were taken with f8at 1/125 second exposure.

A. pT7BPI-F (+) contains the full-length BPI Protein sequence (includingthe signal sequence) placed in the correct orientation behind the T7promoter for expression.

B. pT7BPI-F (-) contains the full-length BPI Protein sequence (includingthe signal sequence) placed in the incorrect orientation behind the T7promoter (resulting protein is a fusion protein with the 260 amino acidleader peptide of T7 gene 10).

C. pT7BPI-S contains the full-length BPI Protein sequence (without thesignal sequence) placed in the correct orientation behind the T7promoter for expression.

D. pT7212-F contains the proline-212 truncated BPI Protein sequence(including the signal sequence) placed in the correct orientation behindthe T7 promoter for expression.

E. pT7212-S contains the proline-212 truncated BPI Protein sequence(without the signal sequence) placed in the correct orientation behindthe T7 promoter for expression.

FIG. 2: Schematic of the pT7BPI Protein plasmid construct.

FIG. 3: Standard curve showing BPI Protein activity in ELISA Assay.

FIG. 4: BPI Protein Sandwich ELISA±endotoxin±Polymyxin B. The protocolis as follows: BPI Protein was performed in the presence and absence ofl μg/ml of polymyxin B sulfate and the presence or absence of l μg/ml E.coli 0111 B4 endotoxin using PBS+1% BSA as diluent.

FIG. 5: Schematic drawing of cDNA encoding BPI.

FIG. 6: A nucleotide (SEQ ID NO:2) and amino acid (SEQ ID NO:1) sequenceof BPI Protein mutagenic primer 25 kDa Pro 212 TGA which is a C-terminaltruncation of BPI Protein.

FIG. 7: A nucleotide (SEQ ID NO:6) and amino acid (SEQ ID NO:5) sequenceof BPI Protein mutagenic primer 38 kDa Pro 337 TGA which is a C-terminaltruncation of BPI Protein.

FIG. 8: A nucleotide (SEQ ID NO:10) and amino acid (SEQ ID NO:9)sequence of BPI Protein mutagenic primer: Preferred ATG 5' HindIII whichis a C-terminal truncation of BPI Protein.

FIG. 9: A schematic drawing of pSVBPIMDH.

FIG. 10A-10B: A schematic drawing of pAc373.

FIG. 11: SDS-PAGE analysis of (1) nBPI Protein (Lot No. #148104), (2)rBPI Protein (Lot No.#148159), and (3) rBPI Protein (Lot No. #148179).

FIG. 12A-12D: cDNA sequence (SEQ ID NO:14) of BPI.

FIG. 13: Protein sequence (SEQ ID NO:15) for p337.

FIG. 14: Protein sequence (SEQ ID NO:16)for p212.

FIG. 15: Line graph showing BPI efficacy using neutropenic rat models.

FIG. 16: Bar graph showing BPI efficacy in vivo.

FIG. 17: Bar graph showing BPI efficacy.

FIG. 16: Line graph showing BPI serum half life.

FIG. 19: Line graph showing BPI binding to endotoxin. BPI binding wasassayed on endotoxin coated wells which were treated with varyingconcentrations of polymyxin B sulfate. Results show absorbance (O.D.405) for buffer control (closed circles). 10 μg/ml polymyxin B (opencircles). 100 μg/ml polymyxin B (closed triangles). 1 mg/ml polymyxin B(open triangles). Data is represented as the mean±SK of quadruplicatevalues.

FIG. 20: Line graph showing BPI endotoxin binding. BPI was diluted inbuffer (closed circles) or neat plasma (open circles) and assayed forendotoxin binding.

FIG. 21: Line graph showing BPI endotoxin binding. BPI was diluted inincreasing concentrations (expressed as ionic strength, mμ) of NaCl(closed circles), MgCl₂ (open circles), or CACl₂ (closed triangles), andassayed for endotoxin binding as described.

FIG. 22: A schematic diagram showing the role of BPI and LBP inregulating endotoxin activity.

FIG. 23: A biologically active variant designated LBP/BPI Chimera (SEQID NO:17).

FIG. 24: A biologically active variant designated CHO-BPI (SEQ IDNO:18).

FIG. 25: A biologically active variant designated BPI (DP linkage) (SEQID NO:19).

FIG. 26: A construct (SEQ ID NO:27) for making biologically activevariants of BPI.

DETAILED DESCRIPTION OF THE INVENTION

As used in this application, the following words or phrases have themeanings specified.

As used herein, "BPI" means a native or naturally occurring biologicallyactive human 57 kd protein which binds to the outer membrane ofsusceptible gram negative bacteria.

As used herein, "biologically active polypeptide fragment of BPI" meansa polypeptide of molecular weight less than 57 kd, having the biologicalactivity of, and an amino acid sequence present within, BPI.

As used herein, "biologically active polypeptide analogs of BPI" means apolypeptide which has substantially the same amino acid sequence as, andthe biological activity of, BPI. Biologically active polypeptide analogsof BPI include polypeptide, the sequence of which varies from thesequence of BPI by a changed amino acid within the BPI sequence, e.g. amutation, or by the addition of one or more amino acids at the amino- orcarboxy- terminus, or both, of the BPI sequence.

As used herein, "biologically active variant of BPI" means a polypeptidethat (1) includes a portion of the amino acid sequence which is presentwithin BPI and an amino acid sequence which is not present within BPI,and (2) has substantially the same biological activity, i.e.endotoxin-neutralizing activity, as BPI.

As used herein, "recombinant" means a polypeptide produced by geneticengineering methods. Thus, each of BPI, biologically active polypeptidefragments of BPI, biologically active polypeptide analogs of BPI, andbiologically active variants of BPI may be recombinant. However, in thecontext of this application, BPI is not the same as recombinant BPI, thelatter differing in some molecular characteristic from the native ornaturally occurring polypeptide, e.g. in glycosylation pattern.

As used herein, BPI Protein means (1) BPI, (2) a biologically activefragment of BPI, (3) a biologically active polypeptide analog of BPI, or(4) a biologically active variant of BPI, each of which may be eitherrecombinant or nonrecombinant.

The present invention provides a composition comprising a BPI Proteinand an anionic compound which composition exhibits (1) no bactericidalactivity and (2) endotoxin neutralizing activity.

In accordance with the practice of this invention, the anionic compoundcould be a protein, a proteoglycan (for example heparin) or a syntheticpolymer (for example dextran sulfate or polyglutamic acid). Preferably,the anionic compound is a protein such as serum albumin.

This invention also provides a biologically active variant of BPI which(1) specifically binds to endotoxin, (2) competes with BPI Protein forbinding to endotoxin, and (3) inhibits endotoxin-induced lethality.

As used in this application the term "endotoxin" means a bacterial toxinwhich is pyrogenic.

One example of a biologically active fragment of BPI is shown in FIG. 13(SEQ ID NO:15). Another example of a biologically active fragment of BPIis shown in FIG. 14 (SEQ ID NO:16).

Additionally, examples of a biologically active variant of BPI is shownin FIG. 23 (SEQ ID NO:17). Another example of a biologically activevariant of BPI is shown in FIG. 24 (SEQ ID NO:18). Further, yet anotherexample of a biologically active variant of BPI is shown in FIG. 25 (SEQID NO:19).

The present invention further provides a method for producing andsecreting a recombinant BPI Protein from a cell. This method comprises(a) constructing a vector comprising DNA encoding BPI Protein; (b)transfecting the cell with the vector; and (c) culturing the cell sotransfected in culture medium under conditions such that BPI Protein issecreted. In accordance with this invention, the vector furthercomprises a signal sequence.

In accordance with this method, mammalian cells are preferred. Examplesof a mammalian cell includes, but is not limited to, HeLa, CHO, DUX B11,Sp2/0, W138, DHK, HEPG2, and COS-1 cells.

This invention also provides a BPI Protein produced by theabove-described method. In one embodiment the BPI Protein is arecombinant BPI Protein designated 148159 rBPI protein shown in FIG. 11.Additionally, the invention provides a recombinant BPI proteindesignated as 148179 rBPI protein shown in FIG. 11.

Interestingly, recombinant BPI Protein produced in mammalian cells suchas Chinese hamster ovary (CHO) cells exhibit a slightly alteredmigration pattern on sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) indicating that the molecule may also beprocessed differently in mammalian cells than in neutrophils or HL60cells. Such processing may be either responsible for, or a result of,the molecule being secreted rather than packaged into granule membranes.

This invention also provides a glycosylated BPI Protein.

In accordance with the above described method, the BPI Protein sosecreted may be a full length soluble BPI Protein.

Also, the present invention provides a method for producing arecombinant BPI Protein from a bacterial cell. This method comprises (a)constructing a vector without a signal sequence and comprising DNAencoding BPI Protein; (b) transfecting the bacterial cell with thevector; and (c) culturing the bacterial cell so transfected in culturemedium under conditions such that recombinant BPI Protein is produced.An example of a bacterial cell includes but is not limited to E. coli.

BPI Protein has been shown to be toxic to bacteria, however, the toxiceffects of the BPI Protein so produced against bacteria may be overcomeby deleting the normal leader sequence in the vector comprising the BPIprotein cDNA.

Apparently, when the signal sequence is included in the expressionplasmid as provided in a full length clone and reported by Gray et al.((1989) Journ. of Biol. Chem., 2:9505) no bacterial colonies areobtained, whereas, numerous colonies can be obtained if the signalsequence is deleted. Further, the method described hereinabove providesfor expression of full length BPI Protein in a nonglycosylated form. Theinvention further provides for a nonglycosylated form of BPI Proteinwhich is free from glycosylated BPI Protein.

The subject invention further provides a method for producing arecombinant BPI Protein from an insect cell. This method comprises (a)constructing a vector without a signal sequence and comprising DNAencoding BPI Protein; (b) transfecting the insect cell with the vector;and (c) culturing the insect cell so transfected in culture medium underconditions such that BPI Protein is secreted.

In one example of the above-described method, insect cells function ashosts for a baculovirus vector containing a sequence encoding the BPIProtein. Also, BPI protein derived from insect cells exhibit a differentmigration pattern on SDS-PAGE than that derived from either mammaliancells or the BPI protein found naturally-occurring in neutrophils. Thus,the invention provides for a new molecular species of BPI Protein asprocessed by baculovirus infected insect cells.

Further, this invention provides a biologically active variant of BPIproduced by the above-described method.

Also, this invention provides a method for determining the amount ofendotoxin in a sample from a subject which comprises contacting thesample with a BPI Protein under conditions such that an endotoxin-BPIProtein complex is formed, detecting the amount of the complex so formedthereby determining the amount of endotoxin in the sample.

Additionally, the present invention provides a method for determiningthe amount of endotoxin in a sample containing bound and unboundendotoxin from a subject. This method comprises (a) treating the sampleso as to denature any endotoxin binding protein to which the endotoxinmay be bound thereby obtaining unbound endotoxin; (b) contacting thetreated sample with a BPI Protein under conditions such that the BPIProtein binds to unbound endotoxin of step (a) so that a endotoxin-BPIProtein complex is formed; (c) detecting the amount of the complex soformed thereby determining the amount of endotoxin in the sample.

In accordance with the invention, denaturation in step (a) may beeffected using an elevated temperature. For example, the elevatedtemperature may be 95° degrees centigrade. Alternatively, denaturationmay be effected with an acid.

This present invention also provides a method of detecting endotoxin ina sample which comprises contacting the sample with a BPI Protein suchthat the endotoxin binds to the BPI Protein and forms a complextherewith; and detecting such complex.

In one example of the invention, the sample containing endotoxin istransferred onto a suitable support under conditions permittingendotoxin in the sample to attach to the support prior to contacting thesample with BPI Protein labeled with a detectable moiety.

This invention further provides a method for diagnosing endotoxemia in asubject which comprises obtaining from the subject a biological fluidsample, detecting endotoxin in such sample using the above-describedmethod and thereby diagnosing such disorder. The sample may be acellular sample. Alternatively, the sample may be a biological fluidsample such as serum, urine, blood, a tissue extract, or sputum.

In accordance with the practice of the invention, the BPI Protein may belabeled with a fluorescent label and detection may be effected by afluorometer. Alternatively, the BPI Protein may be labeled with aradioactive label and detection may be effected by a radiograph.Further, the BPI Protein may be labeled with an enzyme and detection maybe effected by a spectrophotometer.

The present invention further provides a method for coating a surgicaltool with a BPI Protein so that the BPI Protein will complex withendotoxin which method comprises attaching BPI Protein onto a surface ofthe tool which surface is designed for contact with a biological sample.

Also, this invention provides a method for coating an implantable,invasive device with a BPI Protein so that it will form a complex withendotoxin which method comprises attaching BPI Protein onto a surface ofthe device which surface is designed for contact with a biologicalsample.

In accordance with the practice of the present invention, the biologicalsample may be blood. Alternatively, the biological sample may be atissue sample. Further, the biological sample may be a muscle sample.Also, the biological sample may be cartilage. Additionally, thebiological sample may be bone.

Also, in accordance with the practice of this invention, the surgicaltool may be a catheter tubing.

Alternatively, the surgical tool may be a surgical staple.

Further, in accordance with the practice of this invention, the devicemay be a surgical implant.

The present invention further provides a method for decontaminating afluid containing endotoxin prior to administration of the fluid into asubject which comprises contacting the fluid with BPI Protein prior toadministration, under conditions such that endotoxin forms a complexwith BPI Protein, thereby decontaminating the fluid. The fluid may beblood, plasma, blood serum, an isotonic solution, a pharmaceuticalagent, a cell culture reagent, or bone marrow.

This invention also provides a kit for detecting the presence of BPIProtein in a biological fluid sample which comprises (a) polymyxin B inan assay buffer which binds unbound endotoxin molecules; (b) a firstantibody attached to a surface area containing the assay buffer, whichantibody (1) binds to a portion of active BPI Protein and (2) does notcompete with BPI Protein for an endotoxin binding domain; and (c) asecond antibody labeled with a detectable moiety which antibody (1) doesnot compete with the first antibody for BPI Protein binding and (2)specifically binds to BPI Protein at or near the endotoxin binding site,so that when the biological fluid sample is contacted with the first andsecond antibody an active BPI Protein contained in the biological fluidsample is bound by the first and second antibody thus forming a firstantibody-BPI Protein-second antibody complex, detecting such complex,and thereby detecting BPI Protein in the biological fluid sample.

Also, this invention provides a kit for determining the amount of BPIProtein in a biological fluid sample which comprises (a) polymyxin B inan assay buffer which binds unbound endotoxin molecules; (b) a firstantibody attached to a surface area containing the assay buffer, whichantibody (1) binds to a portion of active BPI Protein and (2) does notcompete with BPI Protein for an endotoxin binding domain; and (c) asecond antibody labeled with a detectable moiety which antibody (1) doesnot compete with the first antibody for BPI Protein binding and (2)specifically binds to BPI Protein at or near the endotoxin binding site,so that when the biological fluid sample is contacted with the first andsecond antibody an active BPI Protein contained in the biological fluidsample is bound by the first and second antibody thus forming a firstantibody-active BPI Protein-second antibody complex, detecting suchcomplex, and determining the amount of active BPI Protein in thebiological fluid sample.

Additionally, this invention provides a method for preventingendotoxemia in a subject which comprises administering to the subject anamount of a BPI Protein effective to bind to endotoxin so as to preventendotoxemia in the subject.

The present invention provides a method for treating a subject sufferingfrom endotoxemia which comprises administering to the subject an amountof a BPI Protein effective to bind endotoxin so as to treat the subjectsuffering from endotoxemia.

In accordance with the practice of the invention, the effective amountof the BPI Protein for preventing endotoxemia or treating a subjectsuffering from endotoxemia may be between about 0.1 and about 10 mg/kgbody weight of subject. Also, the effective amount may be an amountbetween about 1 and about 10 mg/kg body weight of subject.

This invention is illustrated in the Experimental Details section whichfollows. This section is set forth to aid an understand of the inventionbut is not intended to, and should not be construed to, limit in any waythe invention as set forth in the claims which follow.

EXPERIMENTAL DETAILS Example 1

Materials and Methods

Reagents and Solutions: endotoxin from E. coli 0111:B4 and from S.typhimurium RE mutant were purchased from RIBI Immunochem Research,Inc., Hamilton, MT. FMLP, cytochalasin B and polymyxin B sulfate (7900U/mg) was purchased from Sigma Chemical Co., St. Louis, Mo. Naturalhuman tumor necrosis factor was purchased from Endogen Inc., Boston,Mass. HBSS without calcium and magnesium and RPMI 1640 were purchasedfrom Gibco BRL, Grand Island, N.Y.

BPI Purification: BPI was purified from neutrophil granule preparationsas previously described (Marra, M.N. et al. J. Immunol. 144: 662, 1990)with the exception that the purification was performed under rigorouslypyrogen-free conditions using new, pyrogen-free columns andde-pyrogenated buffers. Buffers were deyprogenated using a Pyrosartfilter (Sartorius Filters, Hayward, Calif.). Purification of BPI underthese conditions resulted in material with approximately four-foldgreater activity for neutralizing endotoxin-mediated neutrophilstimulation than previously reported (Marra, M. N. et al. J. Immunol.144: 662, 1990).

Immunoaffinity purification of anti-BPI antibodies: Sera was collectedfrom rabbits immunized with a 20 amino acid peptide corresponding to theN-Terminal 20 amino acids of the BPI molecule (BPI peptide 1-20). TheIgG fraction of pooled sera was purified using Protein A Sepharose(Pharmacia, Piscataway, N.J.). Specific anti-peptide IgG was purifiedfrom this fraction using BPI peptide b 1-20 coupled to activated CNBrSepharose (Pharmacia). Bound IgG was collected and pooled, and theadsorbed IgG was further depleted of residual specific antibody bypassing over the peptide column three additional times to generateimmunoadsorbed negative control. Antibody concentration was determinedby optical density at 280 nm. Immunoaffinity purified and adsorbed IgGwere tested for specificity by Western blotting. No activity wasobserved in the immunoadsorbed control IgG, even at concentrations 10³-fold greater than that utilized for the immunoaffinity purifiedantibody.

Endotoxin Binding Assay: BPI binding to endotoxin immobilized onmicrotiter plates was performed using a modified procedure described byTobias, P. S. et al. J. Biol. Chem. 264:10867, 1989. Briefly, Immulon 296 well microtiter plates (Dynatech Biotechnology Products, Chantilly,Va.) were coated with 4 μg/well glycolipid from Salmonella typhimuriumRE mutant in 50 mM borate pH 9+20 mM EDTA overnight at 37° C. Plateswere then washed extensively under running distilled deionized water,then dried at 37° C. Assay plates were blocked for 30 minutes at 37° C.with 5 mg/ml very low endotoxin BSA (Sigma, St. Louis, Mo.) prepared inpyrogen-free PBS. Plates were flicked, and in some experiments polymyxinB was added to the wells and incubated for an additional 30 minutes at37° C. Plates were flicked again, and BPI samples were added. Allbuffers containing BPI or polymyxin B were prepared in pyrogen-free PBS.BPI samples diluted in pyrogen free buffer, or in some experiments,serum or plasma from normal human volunteers, were incubated for 3 hoursat 37° C. with shaking. The plates were washed with PBS containing 1mg/ml pyrogen free BSA, then developed, using rabbit polyclonal anti-BPIpeptide IgG antibody as described followed by goat-anti-rabbitIgG-alkaline phosphatase conjugate (Gibco BRL Life Technologies, Inc.,Grand Island, N.Y.). Absorbances were read at 405 nm on a Vmax kineticmicroplate reader (Molecular Devices Inc., Menlo Park, Calif.).

EPI inhibition of endotoxin mediated TNF induction by human adherentmononuclear cells: Blood collected in acid citrate dextrose containingvacutainer tubes (Becton Dickinson, Rutherford, N.J.) was diluted inHank's balanced salt solution (HBSS) minus Ca² +and Mg²⁺. Mononuclearcells were separated using Ficol-Paque (Pharmacia Inc., Piscataway,N.J.), collected and washed three time in HBSS, and the proportion ofmonocytes was estimated by microscopic examination. Cells were broughtup to an appropriate volume of RPMI 1640 with glutamine and antibioticsand without serum to give approximately 1×10⁶ monocytes/ml. Cells wereplated into 96 well flat bottom tissue culture plates (Costar,Cambridge, Mass.), 200 μl/well, and incubated for 2 hours at 37° C. in ahumidified incubator with 7% O₂. Cells were then washed three times inwarm RPMI 1640 without serum. After the last wash was aspirated, 200μl/well RPMI 1640 with 10% autologous heat inactivated serum was added.To each well was then added the 22 μl of 10X solution of E. coliEndotoxin preincubated in buffer, polymyxin B, or BPI. Cells wereincubated with the endotoxin mixture for 4 hours at 37° C., then thesupernatants were collected and assayed for TNFA antigen by ELISA(Endogen Inc., Boston, Mass.).

Inhibition of endotoxin-induced TNFA secretion by murinebroncheoalveolar macrophages: Normal anesthetized Swiss-Webster micewere challenged by the intranasal route with long E. coli 0111:B4endotoxin (List, Campbell, Calif.). Twenty minutes before challenge,anesthetized mice were treated by the intranasal route with 50 μlsaline, BPI or polymyxin B solution. At one hour after endotoxinchallenge, mice were re-anesthetized, and 0.7 ml of saline containing 1%human serum albumin was added to the lungs via the trachea. The lungswere gently kneaded. A 0.5 ml volume bronchoalveolar lavage (BAL) fluidwas aspirated, cells were pelleted by centrifugation, and the BAL samplewas stored at -70° C. The TNFα level in the BAL fluid was determined bymeasuring cytotoxicity towards WEHI clone 13 mouse fibrosarcoma cells.Human rTNFα (Chiron, Emeryville, Calif.) was used as the standard.

RESULTS

BPI binds to bacterial lipopolysaccharide: Binding of BPI to endotoxinwas demonstrated using a modified ELISA protocol to detect BPI bound toimmobilized S. typhimurium Re endotoxin as described in Methods above.BPI had bound endotoxin in a concentration dependent manner and bindingwas inhibited by polymyxin B/ suggesting that BPI binds at or near lipidA (FIG. 19). Significant binding of BPI to endotoxin was retained in thepresence of plasma (FIG. 20) or serum, thus indicating that BPI binds toendotoxin in the presence of blood proteins as well as physiologicsalts. This date is consistent with the observation by Mannion, B. A. etal. (J. Clin, Invest. 86:631 1990) that BPI binds to bacteria in thepresence of serum albumin, although under these conditions BPI is notbactericidal. Also, concentrations of Ca²⁺ and Mg²⁺ which can rescuebacteria from the lethal actions of BPI (20-80 mM) do not significantlyreduce binding of BPI to endotoxin (FIG. 21).

BPI blocks endotoxin-mediated TNF secretion in vitro: Release in TNF inresponse to endotoxin in vivo may play an important role in pathogenesisof endotoxic shock. To investigate the role of BPI in regulatingendotoxin-mediated TNF secretion, we measured TNF secretion by humanadherent peripheral blood mononuclear cells in response to endotoxin andto endotoxin preincubated with BPI (Table 1). BPI specifically preventedendotoxinstimulated TNF secretion by these cells in a concentrationdependent manner. In addition, inhibition by BPI could be overcome by alarge excess of endotoxin (100-1000 ng/ml) or 0.1% killed S. aureus.indicating that BPI did not interfere with monocyte function but ratherblocked specific activation of monocytes by endotoxin.

                  TABLE 1                                                         ______________________________________                                        Inhibition of endotoxin-Induced TNF Production by BPI                         TNF (pg/ml)                                                                   Endotoxin                                                                             Buffer    Polymyxin  BPI    BPI                                       ng/ml   Control   1.0 μg/ml                                                                             0.4 μg/ml                                                                         0.1 μg/ml                              ______________________________________                                        100     823 ± 67                                                                             400 ± 148                                                                             530 ± 16                                                                          746 ± 48                               10       756 ± 116                                                                           76 ± 25 60 ± 9                                                                            182 ± 42                               1       598 ± 89                                                                             0          0      0                                         ______________________________________                                    

Human peripheral blood mononuclear cells were stimulated with E. coli0111:B4 endotoxin wh ich had been preincubated for 30 minutes at 37° C.with buffer, BPI or polymyxin B. Supernatants were harvested four hoursafter endotoxin mixtures were added. Secretion of TNFA was quantitatedby ELISA.

BPI blocks in vivo pyrogenicity of endotoxin: Cytokines released inresponse to experimental endotoxin infusion cause physiologic changesincluding fever induction. We studied the effects of BPI on endotoxinpyrogenicity by injecting rabbits with endotoxin or endotoxinpreincubated with BPI. Resulting changes in temperature were monitoredat three one-hour intervals post injection. The greatest temperatureincrease was used to calculate Σ(ΔT) for the three animals test in eachgroup. A value of ≧1.4° C. is considered pyrogenic (U.S. PharmacopealConvention, Inc., 1990 Rockville, Md., Test 151, p. 1515). While a totaltemperature rise of 3.9° C. was observed in the group injected with400EU of FDA reference standard endotoxin alone, endotoxin pre-treatedwith 2 μg BPI was not pyrogenic, showing a total temperature rise ofonly 1.1° C. No response was observed in buffer treated control animalsor BPI treated animals.

BPI blocks endotoxin-mediated TNF secretion in vivo: To determinewhether BPI could inhibit endotoxin-mediated TNFα secretion in vivo. wetested BPI neutralization of endotoxin in the murine lung.Administration of BPI into the lung twenty minutes prior to endotoxinchallenge significantly reduced the amount of TNF secreted intobronchoalveolar lavage fluid by alveolar macrophages (Table 2). Four outof five saline treated mice had TNFA levels greater than 1,000 pg/ml,versus one of five for BPI. Overall, BPI reduces endotoxin-mediated TNFαsecretion by murine lung alveolar macrophages by 8.2-fold. Relative tothe saline control, reduction of TNF secretion by BPI was significant(using the Student's t-test) at the p<0.05 level. (Geometric mean±SD ofsaline control: 3.364±0.402, BPI treated group=2.109±0.764). Polymyxin Bwas slightly more effective in reducing TNFα secretion relative to thesaline control (p<0.02) although the dose of PMB was 50-fold greater ona molar basis than that used for BPI. These data indicate the solubleBPI neutralizes endotoxin in vivo.

                  TABLE 2                                                         ______________________________________                                        Effect of BPI on endotoxin-Mediated TNF Secretion by                          Murine BAL                                                                    TNF (pg/ml)                                                                                          BPI      Polymyxin B                                             Saline       0.86 μg                                                                             1.0 μg                                     Mouse     Control      (15 pmol)                                                                              (782 pmol)                                    ______________________________________                                        1         1200         15       74                                            2         675          63       50                                            3         5560         425      132                                           4         2800         67       370                                           5         5250         1310     640                                           Mean ± SD                                                                            3097 ± 2250                                                                             376 ± 547                                                                           253 ± 251                                  ______________________________________                                    

Normal anesthetized mice were challenged by the intranasal route with 10ng E. coli 0111:B4 endotoxin. Twenty minutes before challenge,anesthetized mice were treated by the intranasal route with 50 μlsaline, BPI or polymyxin B solution. Bronchoalveolar lavage (BAL) fluidwas assayed for TNFα by measuring cytotoxicity towards WEHI as clone 13mouse fibrosarcoma cells. Human rTNFα was used at the standard.

Our data show that BPI specifically prevented endotoxin-stimulated TNFsecretion in vitro by human adherent mononuclear cells in aconcentration dependent manner. Inhibition of endotoxin-induced TNFsecretion distinguishes BPI from LBP. LBP, a 60 kDa acute phase proteinsynthesized by hepatocytes, has 44% amino acid sequence homology to BPIand binds to endotoxin in vivo and in vitro (Tobias, P. S., K. Soldau,and R. J. Ulevitch. 1986. Isolation of a lipopolysaccharidebinding acutephase reactant from rabbit serum. J. Exp. Med. 164:777) (Schuman, R. R.,S. R. Leong, G. W. Flaggs, P. W. Gray, S. D. Wright, J. C. Mathison, P.S. Tobias, and R. J. Ulevitch. 1990. Structure and function oflipopolysacchride binding protein. Science. 249:1429). Despite theirstructural similarities, BPI and LBP are functionally antagonistic.LBP-endotoxin complexes prime neutrophils for the oxidative burstresponse to FMLP and cause accelerated and increased TNF production bymonocytes in vitro (Vosbeeck, K., L. Sklar, H. Muller, C. Lundberg, C.Hanson, K. Arfors, R. Ulevitch, and P. Tobias. 1988. Modulation oflipopolysaccharide (LPS) induced neutrophil priming by an acute phasereactant, lipopolysaccharide binding protein, LBP. Eur. J. Clin Invest.18A50) (Wright. S. D., R. A. Ramos, P. S. Tobias, R. J. Ulevitch, and J.C. Mathison. CD14, a receptor for complexes of lipopolysaccharide (LPS)and LPS binding protein. Science. 249:1931) (Tobias, P. S., J. C.,Mathison, and R. J. Ulevitch. 1988. A family of lipopolysaccharidebinding proteins involved in responses to Gram-negative sepsis. J. Biol.Chem. 263:13479). In contrast, BPI blocks LPS-mediated stimulation ofboth neutrophils (Marra, M. N., C. G. Wilde, J. E. Griffith, J. L.Snable and R. W. Scott. 1990. Bactericidal/permeability increasingprotein has endotoxin-neutralizing activity. J. Immunol. 144:662) andmacrophages in vitro. Since BPI-endotoxin complexes fail to stimulateinflammatory cells in vitro. one would not expect such complexes toelicit a pyrogenic response when administered in vivo. Small quantitiesof endotoxin alone induce a strong pyrogenic response resulting from therelease of endogenous pyrogens such as TNF, IL-1, and gamma IFN (Farley,M. M., W. M. Shafer, and J. K. Spitznagel. 1988. Lipopolysaccharidestructure determines ionic and hydrophobic binding of a cationicantimicrobial neutrophil granule protein. Infect. Immun. 56:1589).Rabbits are exquisitely sensitive to trace quantities of endotoxin, andrespond with a dose dependent and reproducible elevation of coretemperature. In complex with BPI, endotoxin was unable to stimulate apyrogenic response in rabbits. Thus, BPI is an effective inhibitor ofendotoxin in vivo presumably a result of BPI blocking endotoxin-mediatedcytokine secretion.

The bactericidal and permeability increasing activities of BPI in vitroare associated with the N-terminal half of the molecule which sharesextensive homology with LBP (Schuman, R. R., S. R. Leong, G. W. Flaggs,P. W. Gray, S. D. Wright, J. C. Mathison P. S. Tobias, and R. J.Ulevitch. 1990. Structure and function of lipopolysaccharide bindingprotein. Science. 249:1429). No function has been ascribed to thecarboxy-terminal region, other than a membrane spanning domain. Gray andcolleagues (Gray, P. W., G. Flaggs, S. R. Leong, R. J. Gumina, J. Weiss,C. E. Ooi, and P. Elsbach. 1989. Cloning of the cDNA of a humanneutrophil bactericidal protein. Structural and functional correlations.J. Biol. Chem. 264:9505) suggest that the carboxy-terminal half of BPIis associated with the azurophil granule membrane. In their model, whenneutrophils are stimulated, proteolytic enzymes such as elastase cleavethe molecule releasing the active, bactericidal N-terminal half into thephagolysosome. Several lines of evidence, however, argue against BPI asan integral membrane protein. BPI can be extracted from isolatedazurophil granules in the absence of detergents. BPI is soluble inaqueous solutions and soluble BPI is active in tests for both endotoxinbinding and inhibition. Also, BPI is released by FMLP/cytochalasin Bstimulated neutrophils (71% of total cellular BPI) as a full-length,protein, arguing against release of the N-terminus by neutrophilproteases upon degranulation.

In vivo, BPI likely functions to suppress endotoxin toxicity and not asa bactericidal protein. Endotoxin binding proteins such as LBP and BPImay function respectively as a receptor/receptor-antagonist system toregulate the host response to endotoxin (FIG. 22). LBP acts as a solublereceptor for endotoxin and amplifies the effects of endotoxin on bothneutrophils and macrophages. The ability of BPI to limit the hostresponse to endotoxin indicates that BPI may have an important role inblocking lethal effects of endotoxin in vivo. Preliminary results inanimals (see Example 4) show that treatment with recombinant BPImarkedly reduces endotoxin-induced lethality. Thus, use of BPI toneutralize endotoxin, in conjunction with conventional antibiotics tolimit bacterial growth, may be a useful therapy against endotoxic shock.

EXAMPLE 2

Expression of BPI Proteins and BPI-Truncated Forms

A. GENETICALLY ENGINEERED MAMMALIAN CELLS EXPRESS BPI

In order to produce BPI protein and/or BPI protein variants in mammaliancells, the cDNA sequences must be inserted into a suitable plasmidvector. A suitable vector for such an application is pSV-1, whichcontains the origin of replication and early and late promoters of SV40,followed by multiple insert cloning sites, followed by the terminationsequences from the hepatitis B surface antigen gene. Also containedwithin the plasmid are an origin of bacterial DNA replication, and thegenes encoding ampicillin resistance and dihydrofolate reductase.Similar vectors have been used to express other foreign genes (McGrogan,et.al. Biotechnology 6, 172-177). Vector DNA was prepared for acceptanceof BPI protein cDNA sequences by digestion with HindIII and Bam HI, anddephosphorylation with alkaline phosphatase.

Several BPI protein cDNA-containing inserts were prepared for insertioninto pSV-1. First, an insert encoding full-length BPI protein wasprepared by digestion of the parent plasmid with appropriate restrictionenzymes for ex. EcoRI and Bg1 II, yielding two DNA fragments containingportions of the BPI protein coding sequence. These two fragments wereligated together into prepared SV-1, and the recombinant clones obtainedwere screened by restriction enzyme digestion for the presence of thetwo inserts in the proper orientation. Two cDNAs encoding truncatedforms of BPI protein were generated using oligonucleotide-directed DNAamplification of the parent BPI protein insert DNA. The amplifyingoligos were designed to replace codons 212 (oligo 459) (SEQ ID NO:4)(FIG. 6) and 337 (oligo 460) (SEQ ID NO:8) (FIG. 7) with stop codons, inaddition to a BamHI cloning site (FIG. 5). At the 5' end of bothconstructs, oligo 458 (SEQ ID NO:11) was used in the amplifications tocreate a HindIII site immediately upstream of the translational startcodon ATG (FIG. 8). Thus, three BPI-encoding inserts were created, eachencoding 55 kDa, 38 kDa, and 25 kDa forms of BPI, and each was ligatedseparately into prepared vector DNA.

Each of the three constructs was verified by restriction digestanalysis, and then prepared in amounts sufficient for transfection intoCHO cell line DUXB11 cells. Transfection was performed using lipofectin,and the resulting transformed cells were selected in the presence ofincreasing amounts of methotrexate using standard protocols (FIG. 3).

Supernatants from either transfected pools or clones derived from thepools were assayed for the presence of endotoxin binding activity byinhibition of TNr release. BPI was negligible in the vast majority ofthe selected cell lines. We found that only cell lines established froma 500 nM methotrexate bulk amplification produced commerciallyreasonable quantities of BPI. Two such cell lines are designated 3A1 and4D6. It was unexpected that only the bulk amplification resulted in suchcell lines.

B. BACULOVIRUS EXPRESSION of rBPI IN INSECT CELLS

Construction of plasmid expression vector: In order to produce BPIprotein and/or BPI protein variants in insect cells, the cDNA sequencemust first be inserted into a suitable plasmid expression vector, suchas pAC373 (FIG. 10). Appropriate restriction sites for this insertionwere created by standard site-directed mutagenesis procedures. Theessential properties of a suitable expression vector include atranscriptional promoter such as the polyhedron gene promoter of pAC373,and flanking homologous sequences to direct recombination into thebaculovirus genome. A polyadenylation signal, such as the one from thepolyhedron gene present in this plasmid vector, may or may not benecessary for expression of the recombinant gene. A marker gene such asthe beta-galactosidase gene of E. coli, juxtaposed to regulatorysequences including a transcriptional promoter and possibly apolyadenylation signal, may be included in the vector but is notessential for BPI protein expression. A typical vector for such purposespAC373, is shown in FIG. 10.

Creation of recombinant baculavirus: A chimeric baculovirus was createdby homologous recombination between the expression plasmid containingthe BPI protein target gene (or truncations thereof derived as describedin Section A) and wild type baculovirus DNA. Plasmid and wild typebaculovirus DNA were co-precipitated by the calcium phosphate techniqueand added to uninfected Spodoptera frugiperda (Sf9) insect cells. Fourto seven days following transfection, cells exhibited a cytopathicmorphology and contained the nuclear occlusion bodies typically producedby viral infection. The cell-free culture media containing both wildtype and recombinant virus was harvested and assayed for BPI activity.

Identification and isolation of chimeric baculavirus: Clonal isolates ofvirus was obtained from this co-transfection stock by plaquepurification on Sf9 cell monolayers overlaid with agarose. Candidateplaques for analysis will be identified by a plaque morphology negativefor occlusion bodies. If the expression plasmid contains a marker genesuch as beta galactosidase, recombinant plaques will be indicated by theblue color produced from a chromogenic substrate such as5-bromo-4-chloryl-3-indolyl-β-D-galactopyranoside (X-gal) in the agaroseplating medium. Picked plaques will be used for inoculation of cells inmultiwell dishes. The resulting cell lysates and infected cellsupernatants can be evaluated for expression of recombinant BPI, usingstandard activity or immunological assays. Positive wells may requireadditional rounds of plaque purification to obtain pure recombinantvirus stocks free from wild type contamination.

Batch production of BPI: Sf9 cells are adapted to growth in serum-free,low protein medium such as ExCell (J. R. Scientific). Cells arecollected from suspension culture by gentle centrifugation andresuspended in fresh medium containing the viral inoculum at aconcentration of ten million cells per ml, using a multiplicity ofinfection of one virus plaque forming unit per cell. After a period oftwo hours, the culture is diluted five fold with fresh medium andincubated two to three days. At the end of that time, the cells werepelleted by centrifugation and the conditioned medium was harvested. BPIprotein was purified from the cell-free supernatant by standard means.

Characterization of insect cell derived BPI: BPI protein produced ininsect cells using a baculovirus expression system is a glycosylatedprotein of approximate molecular weight of 55,000 kd. The N-terminalamino acid sequence is identical to that of mature mammalian cell BPI,indicating correct processing of the signal sequence. The specificactivity of endotoxin binding of recombinant protein wasindistinguishable from BPI.

Construction of pT7BPI protein Plasmids: Oligonucleotides were preparedon an Applied Biosystems 380B DNA Synthesizer for use in oligonucleotidedirected DNA amplification. The oligonucleotides created Nde I and BamHIrestriction sites at the 5' and 3' ends, respectively, of the BPIprotein DNA. In addition, another oligonucleotide containing a BamHIrestriction site was used to create the truncated proline-212 version ofthe BPI protein DNA.

Following the amplification reactions, fragments were purified anddigested with Nde I and BamHI. The plasmid, pGEMEX-1, (available fromPromega) was selected as the vector for the constructions. pGEMEX-1contains a T7 promoter which can be used for the expression ofdownstream sequences when placed into the proper host. The vector wascleaved with BamHI and, following purification, partially digested withNde I to generate a vector with a single Nde I site and a single BamHIsite. The fragments were ligated and transformed into the E. coli strainJM101 using the Hanahan transformation protocol (DNA Cloning Volume I, APractical Approach, Edited by D. M. Glover, IRL Press). The transformedbacteria were plated on LB plates containing carbamicillin and incubatedovernight at 37° C. Resistant colonies were selected and analyzed bypreparing mini-plasmid preparations and digesting with the appropriaterestriction enzymes. Digests were analyzed on both 1% agarose gels and5% polyacrylamide gels.

The expression host, E. coli strain JM109(DE3), was transformed using 1μl of the mini-plasmid preparation and the Hanahan transformationprotocol. JM109(DE3) contains a chromosomal copy of the gene for T7 RNApolymerase which can be induced with IPTG. The transformed bacteria wereplated on LB plates containing carbamicillin and incubated overnight at37° C. Results are shown in FIGS. 1A-1E.

Since the full-length and proline-212 truncated forms of BPI proteincontaining the signal peptide do not give colonies while those formsthat do not contain the signal peptide do give colonies, the BPI proteinwas expressed in an active form and is processed correctly, sending theprotein to the periplasmic space of the bacteria (the location inbacteria that proteins possessing a signal peptide are sent to) wherethe bactericidal activity kills the cell. This also implies that boththe fulllength form and the proline-212 truncated form are active andcapable of bactericidal activity.

Whether the forms of BPI protein which do not contain the signal peptideare active or are prevented from exhibiting their bactericidal activityby being sequestered in the cell (either by the formation of inclusionbodies or by the inability to gain access to the plasma membrane due tothe absence of the signal peptide or both) is not known.

rBPI was purified as follows: Conditioned media containing recombinantBPI (rBPI) was purified to 95% homogeneity in a single step overCM-Sepharose. The CM-Sepharose column (Pharmacia, Piscataway, N.J.) wasfirst washed in five column volumes of 0.5M NaOH followed by rinsingwith pyrogen free buffers or water until no pyrogen could be detected bythe Limulus Amebocyte Lysate Assay (Whittaker, Walkersville, Md.). Thecolumn was then equilibrated in 50 mM Tris pH 7.4. The conditioned mediawas then loaded and the bound protein was eluted in 50 mM Tris 1M NaClpH 7.4. rBPI was concentrated and further purified by loading onto asecond CM-Sepharose column equilibrated in 50 mM Tris pH 7.4 and elutedusing a gradient of 0.0-1.0M NaCl. BPI elutes at approximately 0.75MNaCl. rBPI thus purified appeared as a single band on SDS-polyacrylamidegel electrophoresis and as a single peak on reverse phase HPLC.

EXAMPLE 3

Inhibition of Endotoxin-Induced TNF Production by BPI

Human peripheral blood mononuclear cells were isolated on Ficoll-Paque(Pharmacia) gradients, washed 2X in pyrogen free HBSS (Hazelton), andresuspended at 5×10⁶ /ml in RPMI (Gibco) media without serum. Twohundred μl of this cell suspension was incubated in each well offlat-bottom 96 well tissue culture dishes (Costar) for 2 hours at 37° C.Nonadherent cells were removed by washing 2X with RPMI+10% autologousheat inactivated serum. Adherent mononuclear cells were stimulated withE. coli 0111:B4 endotoxin which had been preincubated for 30 minutes at37° C. with buffer, BPI protein or polymyxin B (Gibco; 7900 U/ml).Supernatants were harvested four hours after endotoxin mixtures wereadded. Secretion of TNFα was quantitated by ELISA (Endogen) (results atTable 3). Several lots of natural and recombinant BPI from CHO cellswere tested.

                                      TABLE 3                                     __________________________________________________________________________    ENDOTOXIN DOSE                                                                10 ng/ml         1 ng/ml       0 ng/ml                                        endotoxin        endotoxin     endotoxin                                      __________________________________________________________________________    BPI   0 7.3                                                                              1.4                                                                              0.3                                                                               0 7.3                                                                              1.4 0.3  0                                             Protein                                                                       (nM):                                                                         Control                                                                            626                                                                              -- -- -- 334                                                                              -- --  --  113                                            78038n                                                                             -- 129                                                                              159                                                                              203                                                                              -- 153                                                                              187 165 --                                             148104n                                                                            --  98                                                                              104                                                                              162                                                                              --  98                                                                              119 162 --                                             148113n                                                                            --  92                                                                              114                                                                              151                                                                              --  71                                                                              129 155 --                                             148159r                                                                            --  82                                                                              158                                                                              155                                                                              --  87                                                                              136 147 --                                             148165r                                                                            -- 124                                                                              128                                                                              138                                                                              -- 116                                                                              129 146 --                                             148179r                                                                            --  85                                                                              139                                                                              134                                                                              --  93                                                                              131 166 --                                             __________________________________________________________________________     n = natural                                                                   r = recombinant                                                          

EXAMPLE 4

The pathophysiologic consequences of gram negative sepsis are primarilymediated by the release of bacterial endotoxin (LPS). Since BPI Proteinhas endotoxin neutralizing activity in vitro, the effects of BPI Proteinin vivo were studied in experimental models of endotoxic shock.

Specifically, in one experiment one group of 8 rats (Sprague Dawleyrats) was given a single, bolus injection of 1 mg BPI Protein per kgbody weight four hours before a single intravenous bolus of 0.5 mg/kgbody weight 0111:B4 endotoxin obtained from Sigma. In the sameexperiment, a second group of 8 rats was given a single bolus injectionof 1 mg BPI Protein per kg body weight simultaneously with a singleintravenous bolus of 0.5 mg/kg body weight 0111:B4 LPS. Further, a thirdgroup of 5 rats was given a single bolus injection of 1 mg BPI Proteinper kg body weight four hours after a single intravenous bolus of 0.5mg/kg body weight 011:B4 LPS. Finally, a fourth group of 10 rats wastreated with endotoxin alone. The rats were observed for 48 hours andthe survival recorded for each group. The results of this experiment areshown in Table 4. Rats to which endotoxin alone was administeredexhibited a mortality rate of 80%. Rats which received both BPI Proteinand endotoxin showed a significantly reduced mortality rate. The resultsset forth in Table 4 establish BPI Protein is useful in vivo both toprevent and to treat disorders associated with the presence ofendotoxin. High dose BPI Protein toxicity studies revealed no evidenceof toxicity when the animals were sacrificed at 7 days. We conclude BPIis a non-toxic naturally occurring protein which binds LPS, inhibitsrelease of TNF and reduces mortality in both endotoxin and GNBexperimental sepsis models (FIG. 17). We believe BPI Protein offers anovel immunotherapeutic approach to the management of septic shock.

                  TABLE 4                                                         ______________________________________                                        INVESTIGATION OF THE POTENTIAL PROTECTIVE EFFICACY                            OF BPI IN THE RAT ENDOTOXIN CHALLENGE MODEL                                   Endotoxin                                                                     Dose     BPI Protein  BPI Protein                                             Survival Dose         Administration Regimen                                                                       %                                        ______________________________________                                        0.5 mg/kg                                                                              --           --             0                                        (2/10)                                                                        0.5 mg/kg                                                                              1 mg/kg      4 hr pre-injection                                                                           5                                        (6/8)                                                                         0.5 mg/kg                                                                              1 mg/kg      simultaneous   5                                        (4/8)                                                                         0.5 mg/kg                                                                              1 mg/kg      4 hr post-injection                                                                          8                                        (4/5)                                                                         ______________________________________                                    

Additionally, in a second experiment with Bactericidal/PermeabilityIncreasing Protein (BPI) neutropenic rats were challenged withPseudomonas (PA1244) during a period of neutropenia. One group of ratswas treated with 10 mg BPI/kg of body weight by intravenousadministration at the onset of fever at day 5 and observed through day11. A second group of rats was treated at the onset of fever with buffercontaining saline at day 5 and observed until day 11. After day 8, therat group treated with buffer was found dead; however, the rat grouptreated with BPI Protein exhibited 60% survival. The rats were observedfor 11 days and the survival recorded for each group. At day 11, noadditional deaths occurred for the rat group treated with BPI. Theresults of this experiment are shown in FIG. 15. FIG. 15 is a line graphshowing that (1) during and after day 8 the rat group treated withbuffer experienced a 100% mortality rate and (2) during and after day 7the rat group treated with BPI Protein exhibited about a 40% mortalityrate. The rats which received BPI Protein showed a significantly reducedmortality rate.

Human BPI Protein at does up to 10 mg/kg intravenously (IV) produced noacute hexatologic, biochemical, or pathologic abnormalities in outbredCD-1 mice or Sprague-Dawley rats (Table 5). Infusion of 1 mg/kg of E.coli 0111:B4 endotoxin IV in 6 CD-1 mice resulted in a 100% (6/6)survival rate in control CD-1 mice. The survival rate in BPI Proteintreated mice infused with 1 mg/kg of E. coli 0111:B4 endotoxin IV at 1mg/kg BPI Protein IV, 2 mg/kg BPI Protein IV, and 10 mg/kg BPI ProteinIV was 100% (4/4), 100% (4/4) and 100 (5/5), respectively.

Infusion of 10 mg/kg of E. coli 0111:B4 endotoxin IV in 6 CD-1 miceresulted in a 17% (1/6) survival rate in control CD-1 mice. The survivalrate in BPI Protein treated mice infused with 1 mg/kg of

                  TABLE 5                                                         ______________________________________                                        BPI PROTEIN PROTECTS AGAINST LETHALITY FROM                                   ENDOTOXIC                                                                     SHOCK (CD-1MICE)                                                                         % SURVIVAL (NO. SURVIVORS/TOTAL NO.                                Endotoxin  ANIMALS TESTED                                                     Challenge                             BPI                                     (E. coli   Control  BPI       BPI     1 0                                     0111:B4)   (Saline) 1 mg/kg IV                                                                              2 mg/kg IV                                                                            mg/kg IV                                ______________________________________                                        * 1 mg/kg IV                                                                             100 (6/6)                                                                              100 (4/4) 100 (4/4)                                                                             100 (5/5)                               * 10 mg/kg IV                                                                             17 (1/6)                                                                               50 (2/4) 100 (4/4)                                                                             100 (5/5)                               * 50 mg/kg IV                                                                             0 (0/6)  25 (2/40  25 (1/4)                                                                             100 (5/5)                               * 100 mg/kg IV                                                                            0 (0/6)  0 (0/4)   0 (0/4)                                                                               80 (4/5)                               * 200 mg/kg IV                                                                            0 (0/6)  0 (0/4)   0 (0/4)                                                                               20 (1/5)                               ______________________________________                                    

E. coli 0111:B4 endotoxin IV at 1 mg/kg BPI Protein IV, 2 mg/kg BPIProtein IV, and 10 mg/kg BPI Protein IV was 50% (2/4), 100% (4/4) and100 (5/5), respectively.

Infusion of 50 mg/kg of E. coli 0111:B4 endotoxin IV in 6 CD-1 miceresulted in a 0% (0/6) survival rate in control CD-1 mice. The survivalrate in BPI Protein treated mice infused with 1 mg/kg of E. coli 0111:B4endotoxin IV at 1 mg/kg BPI Protein IV, 2 mg/kg BPI Protein IV, and 10mg/kg BPI Protein IV was 25% (1/4), 25% (1/4) and 25 (5/5),respectively.

Infusion of 100 mg/kg of E. coli 0111:B4 endotoxin IV in 6 CD1miceresulted in a 0% (0/6) survival rate in control CD1mice. The survivalrate in BPI Protein treated mice infused with 1 mg/kg of E. coli 0111:B4endotoxin IV at 1 mg/kg BPI Protein IV, 2 mg/kg BPI Protein IV, and 10mg/kg BPI Protein IV was 0% (0/4), 0% (0/4) and 80% (4/5), respectively.

Infusion of 200 mg/kg of E. coli 0111:B4 endotoxin IV in 6 CD1miceresulted in a 0% (0/6) survival rate in control CD1mice. The survivalrate in BPI Protein treated mice infused with 1 mg/kg of E. coli 0111:B4endotoxin IV at 1 mg/kg BPI Protein IV, 2 mg/kg BPI Protein IV, and 10mg/kg BPI Protein IV was 0% (0/4), 0% (0/4) and 20% (1/5), respectively.

In conclusion, Table 5 demonstrates that BPI Protein is non-toxic inexperimental animals and provides significant protection from lethalityfollowing endotoxin challenge (FIG. 16). This naturally occurring,neutrophil derived, antimicrobial protein provides a new therapeuticstrategy in the treatment of septic-shock.

Human BPI Protein at does up to 10 mg/kg intravenously (IV) produced noacute hexatologic, biochemical, or pathologic abnormalities in outbredCD1mice (Table 6). Using CD-1 mice, the in vivo efficacy of BPI Proteinagainst endotoxin was tested by infusing 50 mg/kg of E. coli 0111:B4endotoxin IV in 10 mice resulted in a 100% (0/10) survival rate incontrol CD1mice. The survival rate for BPI Protein treated mice infusedwith 50 mg/kg of E. coli 0111:B4 endotoxin IV at 10 mg/kg BPI Protein IVwas 0% (0/10). The p value is p<0.001. Further, 5 mice were infused with50 mg/kg of 055 IV (as control) which resulted in a 0% (0/5) survivalrate. The survival rate for BPI Protein treated mice infused with of 50mg/kg 055 IV at 10 mg/kg BPI Protein IV was 100% (5/5). The p value isp<0.01. Additionally, 5 mice were infused with 25 mg/kg of Rc roughmutant (core glycolipid) IV (as control) which resulted in a 0% (0/5)survival rate. The survival rate for BPI Protein treated mice infusedwith 25 mg/kg of Rc rough mutant (core glycolipid)IV at 10 mg/kg BPIProtein IV was 100% (5/5). The p value is p<0.01. Also, 4 mice wereinfused with 25 mg/kg of Lipid A IV (as control) which resulted in a 0%(0/4) survival rate. The survival rate for BPI Protein treated miceinfused with 25 mg/kg of Lipid A IV at 10 mg/kg BPI Protein IV was 100%(5/5). The p value is p<0.05.

BPI is a non-toxic naturally occurring protein with endotoxinneutralizing activity which reduces mortality in both endotoxic andbacteremic models of sepsis syndrome and may be a usefulimmunotherapeutic approach to the management of the septic shock.

                  TABLE 6                                                         ______________________________________                                        EFFECT OF BPI PROTEIN ON LETHALITY OF VARIOUS                                 ENDOTOXIN PHENOTYPES                                                                       % Survival                                                                    (No. Surviving/No. Animals Tested)                                              BPI                                                            endotoxin Phenotype                                                                          10 mg/kg   Control p Value                                     ______________________________________                                        * 0111:B4      100 (10/10)                                                                              0 (1/10)                                                                              p < 0.001                                   50 mg/kg                                                                      * 055          100 (5/5)  0 (0/5) p = 0.01                                    50 mg/kg                                                                      * Rc Rough mutant                                                                            100 (5/5)  0 (0/5) p = 0.01                                    25 mg/kg                                                                      * Lipid A      100 (5/5)  0 (0/4) p < 0.05                                    25 mg/kg                                                                      ______________________________________                                    

In order to generate a non-glycosylated form of the BPI molecule, the(CHO) cell line which normally expresses glycosylated recombinant BPIProtein (clone 3A1), was grown to confluence in roller bottles (Costar,Cambridge, Mass.) in REM 020 (Hazelton, Inc. Denver, Pa.) containing7.5% dialyzed bovine serum (Gibco)+2 μg/ml tunicamycin (BoehringerMannheim, Indianapolis, Ind.). After 24 hours, the medium was discarded,and replaced with fresh complete medium containing 2 μ/ml tunicamycin.Conditioned medium was collected and replaced every 24 hours for threedays. Non-glycosylated recombinant BPI Protein was purified as describedin Example 3 above for recombinant BPI Protein and further separatedfrom residual glycosylated recombinant BPI Protein by Superose 12(Pharmacia) size exclusion chromatography in 20 mM glycine+100 mM NaClat PH 2. Fractions containing nonglycosylated BPI Protein (identified bypolyacrylamide gel electrophoresis) were pooled.

Glycosylated or nonglycosylated recombinant BPI Protein was injectedinto mice at 10 mg/kg. Blood was collected at the indicated timesthrough the retroorbital plexus. Blood samples were then allowed toclot, the fibrin clot was removed by centrifugation, and the recombinantBPI Protein levels were determined by ELISA assay (results are shown inFIG. 18).

ELISA ASSAY

EQUIPMENT

Immulon-2 96 well plates (Dynatech) 12-channel 50-200 μl pipettor P20,P200, P1000 pipettors Reagent reservoirs (Costar) Racked 1 ml tubes(BioRad) polypropylene 15 ml conical tubes

REAGENTS

SOLUTIONS

25 Mm Borate pH 9.5

Blocking solution=5% BSA (Sigma Fraction V, Low

Endotoxin) in PBS

Wash/Sample Buffer:

    ______________________________________                                        Wash/Sample Buffer:                                                                             50 mM Tris pH 7.4                                                             500 mM NaCl                                                                   1 mg/ml BSA                                                                   0.05% Tween 20                                                                1 μ/ml Polymyxin B Sulfate                                                 (Gibco/BRL, 7900 U/mg)                                      ______________________________________                                    

BPI standard (aliquots stored @-70° C.)

NOTE: BPI standards and samples should be diluted in polypropylene

Standard and sample diluent=appropriate solution for unknowns

(e.g. if testing tissue culture supernatants, use REM+7.5% dFBS)

Substrate Buffer: (makes 500 ml)

24.5 mg MgCl₂

48 ml ethanolamine

bring up to -400 ml with Lab V H₂ O

Adjust to pH 9.8

Bring up to 500 ml with Lab V H₂ O

PNPP substrate tablets (5 mg/tablet: Sigma)

ANTIBODIES

Capture (1st) Antibody (100 μl/well)

A. INVN 1-2 (rabbit polyclonal anti-human BPI Protein) IgG 1 μg/ml, or,

B. MM-1 (rabbit anti N-terminal 20 amino acid BPI peptide) 3 μg/ml.

Reporter (2nd) Antibody

A. INVN 1-2-Biotin (Use @ 1:1000)

B. PIG8 (murine monoclonal anti-BPI which blocks BPI binding to bacteria

Third (developing) reagent

A. Streptavidin/Alkaline Phosphatase (BioRad) (use @ 1:2000)

B. Goat anti-mouse Ig/Alkaline Phosphatase conjugate (BioRad) (use @1:2000)

PROCEDURE

1. COATING PLATES

Note: Coat plates up to 1 month in advance. Store plates at 4° C. untilneeded.

Dilute capture antibody as directed in 25mM Na Borate pH 9.5 (10ml/plate).

Add 100 μl to cach well of 96 well plate (Immulon-2). Incubate overnightat 37° C. Refrigerate until used.

2. BLOCKING

Flick coating antibody out of plates. Add 200 μl 5% BSA in PBS to eachwell. Incubate 2-4 hours 37° C. or overnight at 4° C. Wash 4X with washsolution and blot on paper towels.

3. BPI STANDARDS AND UNKNOWNS STANDARDS

Thaw new standard aliquot (0.5 ml @ 1 mg/ml) every 2 months.

1. Make 1ml stock solution of purified or BPI at 100 ng/ml

2. Make 500 μl of each of the following standard concentrations asfollows:

    ______________________________________                                        μl 100 ng/ml BPI                                                                          μ diluent                                                                           final  BPI! ng/ml                                     ______________________________________                                        150            350      30                                                    100            400      20                                                    75             425      15                                                    50             450      10                                                    40             460      8                                                     25             475      5                                                     10             490      2                                                     0               50      0                                                     ______________________________________                                    

Add 100 μl standard (unknown)/well and incubate at RT for 2-4 hours, orovernight at 4° C.

wash 4X

2nd ANTIBODY

After final wash, blot plate vigorously, and add 100 μl ofINVN1-2-Biotinylated @ 1:1000 (=10 μl in 10 ml of wash/sample buffer) toeach well.

Incubate 37° C. 1 hour

Wash 4X

3rd ANTIBODY

After final wash, blot plate vigorously, and add 100 μl developingreagent to each well.

Incubate 37° C. 30 minutes

Wash 4X

SUBSTRATE

NOTE: Add substrate tablets to substrate buffer just before adding toplate.

After final wash, blot plate vigorously, and add 100 μl substratesolution (2X 5 mg PNPP substrate tablet/10 ml substrate buffer)

Read plate at 405 nm. Keep plate in the dark between readings.

EXAMPLE 5

Biologically Active Variants of BPI: Several classes of variants of BPIwere constructed to alter some of the different properties of the nativemolecule(SEQ ID NOS:13 and 14). In the first type of construct, variantswere designed to extend the molecular half-life in serum. In one of suchconstructs, the single glycosylation site at Ser351 was altered bymaking a single base pair change at position 1175 (FIG. 12) so that itencodes Ala and would not support N-glycosylation (i.e.Ser351->AlaBPI(nonglycosylated) (SEQ ID NO:18) at Table 7). This changewas made by amplifying this particular segment of the molecule by PCRusing amplimers containing the desired sequence, and then replacing thenative segment with the corrected segment by virtue of convenientrestriction sites (the SphI site at base 1202 in this case). Such amolecule was expected to possess similar properties as BPI but may becleared less rapidly by the liver since it would lack the mannoseresidues recognized by hepatic clearance receptors. Other constructswere designed to take advantage of the apparent high stability of LBP, ahomolog of BPI. For instance, the amino-terminal 25 kDa segment(presumably the endotoxin-binding domain) of LBP was combined with thecarboxy-terminal 30 kDa portion of BPI to create a chimeric moleculewith the greater serum half-life of LBP but the functionality of BPI(i.e. LBP25K/BPI30K chimeric (SEQ ID NO:17) at Table 7).

A third type of construct utilized the extraordinary serum stability ofimmunoglobin to extend the stability of BPI. The amino-terminal 25 kDa(LPS-binding) portion of BPI was linked to cDNA encoding the constantdomain of IgG₁. The resultant chimeric molecule could be expected tobind endotoxin and inactivate it like the anti-endotoxin antibodiescurrently under development.

A second class of molecules were designed to enhance the therapeuticindex of BPI Protein. For example, the amino terminal domain of BPIProtein contains a very high proportion of positively-charged residues(approximately 14%). In several of the variants, one or more of theamino terminal cationic residues were changed to neutral or negativelycharged residues by the methods described below. Such redesignedmolecules may prove less disruptive to biological membranes andtherefore be less cationic. Also, the LBP/BPI Protein chimeric moleculedescribed below (i.e. LBP25K/BP130K chimeric at Table 7) may also beless toxic due to the reduced cationicity of the LBP amino terminaldomain relative to BPI.

A third class of variants were intended to increase the affinity,specificity, and/or valency of endotoxin binding to BPI. For example,recombinant BPI Protein containing single base pair changes within the25 kDA portion were produced and tested for their ability to bindendotoxin in vitro. Changing certain key amino acids, particularlycationic residues, may enhance the affinity of BPI Protein for itsligand, i.e. endotoxin. Also, the LBP/BPI Protein chimeric moleculedesignated LBP25K/BPI30K chimeric may have the affinity of LPB forendotoxin, while possessing the functionality of BPI. Other constructsadded a second endotoxin-binding domain to BPI, under the expectationthat it may bind twice the amount of endotoxin per BPI Protein.

A fourth class of mutants were designed to modify the binding affinityof BPI and/or BPI/endotoxin complexes for the receptors with which itnormally interacts to downregulate macrophage activation. Examples ofthis include BPI Protein with single amino acid changes within the 30kDa portion of BPI, created by in vitro mutagenesis as described below.One such mutant designated BPI25K/DP/BPI30K (SEQ ID NO:19) (Table 7)created a variant from which the intact 25 kDA domain could be liberatedby treatment with formic acid.

Methods used to create the biologically active variants of BPI werestandard as practiced in the art. Relevant portions of key moleculeswere recombined to form chimeric molecules through commonly usedmethods. For example, the amino-terminal 25 kDa portion of LBP waslinked to the carboxy-terminal portion of BPI Protein by virtue of anengineered ClaI site within the coding sequence (SEQ ID NO:27), as shownin FIG. 26. Oligonucleotide amplimers (SEQ ID NOS:20-25 and 27)containing cloning sites as well as a segment of DNA sufficient tohybridize to stretches at both ends of the target cDNA (25 bases) weresynthesized chemically by standard methods. These primers were then usedto amplify the desired gene segments by Polymerase Chain Reaction. Theresulting new gene segments were digested with the correspondingrestriction enzyme under standard conditions, and were isolated by gelelectrophoresis. Alternately, similar gene segments were produced bydigestion of the cDNA itself with appropriate restriction enzymes, andfilling in missing gene segments with chemically synthesizedoligonucleotides. These segments of coding sequence were ligatedtogether and cloned into appropriate expression vectors which wouldallow recombinant production of the encoded sequence. Relevantexpression systems for such chimeric molecules include but are notlimited to mammalian cells such as CHO, fungi such as yeast, insectviruses such as baculavirus, and bacteria such as E. coli. For each ofthese systems, a useful expression vector would include antibioticresistance gene such as ampicillin which would allow selection ofrecombinant clones; a second antibiotic resistance gene to allowselection on the host cells, such as neomycin; a bacterial replicationorigin to allow propagation in bacteria; a high level promoter upstreamof the gene insertion site, such as the MMTV, SV40, or metallothioninepromoter for CHO cells, the trp, lac, tac or T7 promoter for bacterialhosts, or the alpha factor, gal, or PGDH promoters in yeast;transcription enhancers for the mammalian hosts, such as the RSVenhancer; and a polyadenylation site, AATAAA, if one does not existwithin the CDNA sequence. Once homogeneous cultures of recombinant cellswere obtained through standard culture methods, large quantities ofrecombinant chimeric molecules were recovered and analyzed from theconditioned medium through standard chromatographic methods.

As examples, three of the constructs described above were constructed invector pMamNeo, a commercially available expression vector (Clontech,Mountain View, Calif.), and used to transform mammalian cell hostDUXB11. After transformation using lipofectin, a commercially availablereagent (BRL/Gibco Gaithersberg, Md.), the cells were cultured instandard tissue culture medium to allow recovery and phenotypicexpression of neomycin resistance. After 24 hours of recovery, theselective agent G418 was added to the medium to select for cellsexpressing the introduced genes. After three weeks of culture inselective media, drug resistant cell pools were obtained and grown toconfluent densities. At this time, media was removed and assayed for thepresence of immunoreactivity to anti-BPI Protein by ELISA, and forbinding to endotoxin prebound to multiwell plates. In some cases, 160 nMdexamethasone was added to the medium to enhance expression because thevector also contained a glucocorticoid binding site in the promoterregion. The levels of BPI Protein produced were monitored in eachsupernatant sample taken, and representative date is shown below:

                  TABLE 7                                                         ______________________________________                                                                      Endotoxin                                                             ELISA   Binding                                         Culture Description   ng/ml   ng/ml                                           ______________________________________                                        A LBP25K/BP130K chimeric                                                                            4.3     7.8                                             B Ser182 -> AlaBPI (nonglycosylated)                                                                8.3     10.8                                            C BP125K/DP/BPI30K    5.8     5.2                                             D BPI                 3.9     3.7                                             ______________________________________                                    

Therefore, these three biologically active variants of BPI were shown tobe produced in CHO hosts, were immunologically crossreactive withanti-BPI Antibody, and able to bind endotoxin at levels similar to BPI.The same vector was transfected into alternate cell host lines to see ofimproved levels could be achieved. Constructs from which largequantities of recombinant protein was desired were also recloned into anamplifyable vector such as pSE, containing the gene encodingdihydrofolate reductase.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 27                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       IleAsnTyrGlyLeuValAlaPro                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TAGAACTATGGTCTGGTGGCACCTTGAGGATCCGCG36                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CGCGGATCCTCAAGGTGCCACCAGACCATA30                                              (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CGCGGATCCTCAAGGTGCCACCAGACCATA30                                              (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ProThrGlyLeuThrPheTyrPro                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CCCACCGGCCTTACCTTCTACCCTTGAGGATCCGCG36                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CGCGGATCCTCAAGGGTAGAAGGTAAGGCC30                                              (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CGCGGATCCTCAAGGGTAGAAGGTAAGGCC30                                              (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       MetArgGluAsnMetArg                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CCCAAGCTTGCCACCATGAGAGAGAACATGGCC33                                           (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CCCAAGCTTGCCACCATGAGAGAGAACATGGCC33                                           (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AAAAAAACCCGAGATCCGCGGATCCTTTCCT31                                             (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 487 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      MetArgGluAsnMetAlaArgGlyProCysAsnAlaProArgTrpVal                              151015                                                                        SerLeuMetValLeuValAlaIleGlyThrAlaValThrAlaAlaVal                              202530                                                                        AsnProGlyValValValArgIleSerGlnLysGlyLeuAspTyrAla                              354045                                                                        SerGlnGlnGlyThrAlaAlaLeuGlnLysGluLeuLysArgIleLys                              505560                                                                        IleProAspTyrSerAspSerPheLysIleLysHisLeuGlyLysGly                              65707580                                                                      HisTyrSerPheTyrSerMetAspIleArgGluPheGlnLeuProSer                              859095                                                                        SerGlnIleSerMetValProAsnValGlyLeuLysPheSerIleSer                              100105110                                                                     AsnAlaAsnIleLysIleSerGlyLysTrpLysAlaGlnLysArgPhe                              115120125                                                                     LeuLysMetSerGlyAsnPheAspLeuSerIleGluGlyMetSerIle                              130135140                                                                     SerAlaAspLeuLysLeuGlySerAsnProThrSerGlyLysProThr                              145150155160                                                                  IleThrCysSerSerCysSerSerHisIleAsnSerValHisValHis                              165170175                                                                     IleSerLysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLys                              180185190                                                                     IleGluSerAlaLeuArgAsnLysMetAsnSerGlnValCysGluLys                              195200205                                                                     ValThrAsnSerValSerSerLysLeuGlnProTyrPheGlnThrLeu                              210215220                                                                     ProValMetThrLysIleAspSerValAlaGlyIleAsnTyrGlyLeu                              225230235240                                                                  ValAlaProProAlaThrThrAlaGluThrLeuAspValGlnMetLys                              245250255                                                                     GlyGluPheTyrSerGluAsnHisHisAsnProProProPheAlaPro                              260265270                                                                     ProValMetGluPheProAlaAlaHisAspArgMetValTyrLeuGly                              275280285                                                                     LeuSerAspTyrPhePheAsnThrAlaGlyLeuValTyrGlnGluAla                              290295300                                                                     GlyValLeuLysMetThrLeuArgAspAspMetIleProLysGluSer                              305310315320                                                                  LysPheArgLeuThrThrLysPhePheGlyThrPheLeuProGluVal                              325330335                                                                     AlaLysLysPheProAsnMetLysIleGlnIleHisValSerAlaSer                              340345350                                                                     ThrProProHisLeuSerValGlnProThrGlyLeuThrPheTyrPro                              355360365                                                                     AlaValAspValGlnAlaPheAlaValLeuProAsnSerSerLeuAla                              370375380                                                                     SerLeuPheLeuIleGlyMetHisThrThrGlySerMetGluValSer                              385390395400                                                                  AlaGluSerAsnArgLeuValGlyGluLeuLysLeuAspArgLeuLeu                              405410415                                                                     LeuGluLeuLysHisSerAsnIleGlyProPheProValGluLeuLeu                              420425430                                                                     GlnAspIleMetAsnTyrIleValProIleLeuValLeuProArgVal                              435440445                                                                     AsnGluLysLeuGlnLysGlyPheProLeuProThrProAlaArgVal                              450455460                                                                     GlnLeuTyrAsnValValLeuGlnProHisGlnAsnPheLeuLeuPhe                              465470475480                                                                  GlyAlaAspValValTyrLys                                                         485                                                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1813 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CAGGCCTTGAGGTTTTGGCAGCTCTGGAGGATGAGAGAGAACATGGCCAGGGGCCCTTGC60                AACGCGCCGAGATGGGTGTCCCTGATGGTGCTCGTCGCCATAGGCACCGCCGTGACAGCG120               GCCGTCAACCCTGGCGTCGTGGTCAGGATCTCCCAGAAGGGCCTGGACTACGCCAGCCAG180               CAGGGGACGGCCGCTCTGCAGAAGGAGCTGAAGAGGATCAAGATTCCTGACTACTCAGAC240               AGCTTTAAGATCAAGCATCTTGGGAAGGGGCATTATAGCTTCTACAGCATGGACATCCGT300               GAATTCCAGCTTCCCAGTTCCCAGATAAGCATGGTGCCCAATGTGGGCCTTAAGTTCTCC360               ATCAGCAACGCCAATATCAAGATCAGCGGGAAATGGAAGGCACAAAAGAGATTCTTAAAA420               ATGAGCGGCAATTTTGACCTGAGCATAGAAGGCATGTCCATTTCGGCTGATCTGAAGCTG480               GGCAGTAACCCCACGTCAGGCAAGCCCACCATCACCTGCTCCAGCTGCAGCAGCCACATC540               AACAGTGTCCACGTGCACATCTCAAAGAGCAAAGTCGGGTGGCTGATCCAACTCTTCCAC600               AAAAAAATTGAGTCTGCGCTTCGAAACAAGATGAACAGCCAGGTCTGCGAGAAAGTGACC660               AATTCTGTATCCTCCAAGCTGCAACCTTATTTCCAGACTCTGCCAGTAATGACCAAAATA720               GATTCTGTGGCTGGAATCAACTATGGTCTGGTGGCACCTCCAGCAACCACGGCTGAGACC780               CTGGATGTACAGATGAAGGGGGAGTTTTACAGTGAGAACCACCACAATCCACCTCCCTTT840               GCTCCACCAGTGATGGAGTTTCCCGCTGCCCATGACCGCATGGTATACCTGGGCCTCTCA900               GACTACTTCTTCAACACAGCCGGGCTTGTATACCAAGAGGCTGGGGTCTTGAAGATGACC960               CTTAGAGATGACATGATTCCAAAGGAGTCCAAATTTCGACTGACAACCAAGTTCTTTGGA1020              ACCTTCCTACCTGAGGTGGCCAAGAAGTTTCCCAACATGAAGATACAGATCCATGTCTCA1080              GCCTCCACCCCGCCACACCTGTCTGTGCAGCCCACCGGCCTTACCTTCTACCCTGCCGTG1140              GATGTCCAGGCCTTTGCCGTCCTCCCCAACTCCTCCCTGGCTTCCCTCTTCCTGATTGGC1200              ATGCACACAACTGGTTCCATGGAGGTCAGCGCCGAGTCCAACAGGCTTGTTGGAGAGCTC1260              AAGCTGGATAGGCTGCTCCTGGAACTGAAGCACTCAAATATTGGCCCCTTCCCGGTTGAA1320              TTGCTGCAGGATATCATGAACTACATTGTACCCATTCTTGTGCTGCCCAGGGTTAACGAG1380              AAACTACAGAAAGGCTTCCCTCTCCCGACGCCGGCCAGAGTCCAGCTCTACAACGTAGTG1440              CTTCAGCCTCACCAGAACTTCCTGCTGTTCGGTGCAGACGTTGTCTATAAATGAAGGCAC1500              CAGGGGTGCCGGGGGCTGTCAGCCGCACCTGTTCCTGATGGGCTGTGGGGCACCGGCTGC1560              CTTTCCCCAGGGAATCCTCTCCAGATCTTAACCAAGAGCCCCTTGCAAACTTCTTCGACT1620              CAGATTCAGAAATGATCTAAACACGAGGAAACATTATTCATTGGAAAAGTGCATGGTGTG1680              TATTTTAGGGATTATGAGCTTCTTTCAAGGGCTAAGGCTGCAGAGATATTTCCTCCAGGA1740              ATCGTGTTTCAATTGTAACCAAGAAATTTCCATTTGTGCTTCATGAAAAAAAACTTCTGG1800              TTTTTTTCATGTG1813                                                             (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 368 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      MetArgGluAsnMetAlaArgGlyProCysAsnAlaProArgTrpVal                              151015                                                                        SerLeuMetValLeuValAlaIleGlyThrAlaValThrAlaAlaVal                              202530                                                                        AsnProGlyValValValArgIleSerGlnLysGlyLeuAspTyrAla                              354045                                                                        SerGlnGlnGlyThrAlaAlaLeuGlnLysGluLeuLysArgIleLys                              505560                                                                        IleProAspTyrSerAspSerPheLysIleLysHisLeuGlyLysGly                              65707580                                                                      HisTyrSerPheTyrSerMetAspIleArgGluPheGlnLeuProSer                              859095                                                                        SerGlnIleSerMetValProAsnValGlyLeuLysPheSerIleSer                              100105110                                                                     AsnAlaAsnIleLysIleSerGlyLysTrpLysAlaGlnLysArgPhe                              115120125                                                                     LeuLysMetSerGlyAsnPheAspLeuSerIleGluGlyMetSerIle                              130135140                                                                     SerAlaAspLeuLysLeuGlySerAsnProThrSerGlyLysProThr                              145150155160                                                                  IleThrCysSerSerCysSerSerHisIleAsnSerValHisValHis                              165170175                                                                     IleSerLysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLys                              180185190                                                                     IleGluSerAlaLeuArgAsnLysMetAsnSerGlnValCysGluLys                              195200205                                                                     ValThrAsnSerValSerSerLysLeuGlnProTyrPheGlnThrLeu                              210215220                                                                     ProValMetThrLysIleAspSerValAlaGlyIleAsnTyrGlyLeu                              225230235240                                                                  ValAlaProProAlaThrThrAlaGluThrLeuAspValGlnMetLys                              245250255                                                                     GlyGluPheTyrSerGluAsnHisHisAsnProProProPheAlaPro                              260265270                                                                     ProValMetGluPheProAlaAlaHisAspArgMetValTyrLeuGly                              275280285                                                                     LeuSerAspTyrPhePheAsnThrAlaGlyLeuValTyrGlnGluAla                              290295300                                                                     GlyValLeuLysMetThrLeuArgAspAspMetIleProLysGluSer                              305310315320                                                                  LysPheArgLeuThrThrLysPhePheGlyThrPheLeuProGluVal                              325330335                                                                     AlaLysLysPheProAsnMetLysIleGlnIleHisValSerAlaSer                              340345350                                                                     ThrProProHisLeuSerValGlnProThrGlyLeuThrPheTyrPro                              355360365                                                                     (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 243 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      MetArgGluAsnMetAlaArgGlyProCysAsnAlaProArgTrpVal                              151015                                                                        SerLeuMetValLeuValAlaIleGlyThrAlaValThrAlaAlaVal                              202530                                                                        AsnProGlyValValValArgIleSerGlnLysGlyLeuAspTyrAla                              354045                                                                        SerGlnGlnGlyThrAlaAlaLeuGlnLysGluLeuLysArgIleLys                              505560                                                                        IleProAspTyrSerAspSerPheLysIleLysHisLeuGlyLysGly                              65707580                                                                      HisTyrSerPheTyrSerMetAspIleArgGluPheGlnLeuProSer                              859095                                                                        SerGlnIleSerMetValProAsnValGlyLeuLysPheSerIleSer                              100105110                                                                     AsnAlaAsnIleLysIleSerGlyLysTrpLysAlaGlnLysArgPhe                              115120125                                                                     LeuLysMetSerGlyAsnPheAspLeuSerIleGluGlyMetSerIle                              130135140                                                                     SerAlaAspLeuLysLeuGlySerAsnProThrSerGlyLysProThr                              145150155160                                                                  IleThrCysSerSerCysSerSerHisIleAsnSerValHisValHis                              165170175                                                                     IleSerLysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLys                              180185190                                                                     IleGluSerAlaLeuArgAsnLysMetAsnSerGlnValCysGluLys                              195200205                                                                     ValThrAsnSerValSerSerLysLeuGlnProTyrPheGlnThrLeu                              210215220                                                                     ProValMetThrLysIleAspSerValAlaGlyIleAsnTyrGlyLeu                              225230235240                                                                  ValAlaPro                                                                     (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 454 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      AlaAsnProGlyLeuValAlaArgIleThrAspLysGlyLeuGlnTyr                              151015                                                                        AlaAlaGlnGluGlyLeuLeuAlaLeuGlnSerGluLeuLeuArgIle                              202530                                                                        ThrLeuProAspPheThrGlyAspLeuArgIleProHisValGlyArg                              354045                                                                        GlyArgTyrGluPheHisSerLeuAsnIleHisSerCysGluLeuLeu                              505560                                                                        HisSerAlaLeuArgProValProGlyGlnGlyLeuSerLeuSerIle                              65707580                                                                      SerAspSerSerIleArgValGlnGlyArgTrpLysValArgLysSer                              859095                                                                        PhePheLysLeuGlnGlySerPheAspValSerValLysGlyIleSer                              100105110                                                                     IleSerValAsnLeuLeuLeuGlySerGluSerSerGlyArgProThr                              115120125                                                                     GlyTyrCysLeuSerCysSerSerAspIleAlaAspValGluValAsp                              130135140                                                                     MetSerGlyAspSerGlyTrpLeuLeuAsnLeuPheHisAsnGlnIle                              145150155160                                                                  GluSerLysPheGlnLysValLeuGluSerArgIleCysGluMetIle                              165170175                                                                     GlnLysSerValSerSerAspLeuGlnProTyrLeuGlnThrLeuPro                              180185190                                                                     ValThrThrGluIleAspSerValAlaGlyIleAsnTyrGlyLeuVal                              195200205                                                                     AlaProProAlaThrThrAlaGluThrLeuAspValGlnMetLysGly                              210215220                                                                     GluPheTyrSerGluAsnHisHisAsnProProProPheAlaProPro                              225230235240                                                                  ValMetGluPheProAlaAlaHisAspArgMetValTyrLeuGlyLeu                              245250255                                                                     SerAspTyrPhePheAsnThrAlaGlyLeuValTyrGlnGluAlaGly                              260265270                                                                     ValLeuLysMetThrLeuArgAspAspMetIleProLysGluSerLys                              275280285                                                                     PheArgLeuThrThrLysPhePheGlyThrPheLeuProGluValAla                              290295300                                                                     LysLysPheProAsnMetLysIleGlnIleHisValSerAlaSerThr                              305310315320                                                                  ProProHisLeuSerValGlnProThrGlyLeuThrPheTyrProAla                              325330335                                                                     ValAspValGlnAlaPheAlaValLeuProAsnSerSerLeuAlaSer                              340345350                                                                     LeuPheLeuIleGlyMetHisThrThrGlySerMetGluValSerAla                              355360365                                                                     GluSerAsnArgLeuValGlyGluLeuLysLeuAspArgLeuLeuLeu                              370375380                                                                     GluLeuLysHisSerAsnIleGlyProPheProValGluLeuLeuGln                              385390395400                                                                  AspIleMetAsnTyrIleValProIleLeuValLeuProArgValAsn                              405410415                                                                     GluLysLeuGlnLysGlyPheProLeuProThrProAlaArgValGln                              420425430                                                                     LeuTyrAsnValValLeuGlnProHisGlnAsnPheLeuLeuPheGly                              435440445                                                                     AlaAspValValTyrLys                                                            450                                                                           (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 456 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      ValAsnProGlyValValValArgIleSerGlnLysGlyLeuAspTyr                              151015                                                                        AlaSerGlnGlnGlyThrAlaAlaLeuGlnLysGluLeuLysArgIle                              202530                                                                        LysIleProAspTyrSerAspSerPheLysIleLysHisLeuGlyLys                              354045                                                                        GlyHisTyrSerPheTyrSerMetAspIleArgGluPheGlnLeuPro                              505560                                                                        SerSerGlnIleSerMetValProAsnValGlyLeuLysPheSerIle                              65707580                                                                      SerAsnAlaAsnIleLysIleSerGlyLysTrpLysAlaGlnLysArg                              859095                                                                        PheLeuLysMetSerGlyAsnPheAspLeuSerIleGluGlyMetSer                              100105110                                                                     IleSerAlaAspLeuLysLeuGlySerAsnProThrSerGlyLysPro                              115120125                                                                     ThrIleThrCysSerSerCysSerSerHisIleAsnSerValHisVal                              130135140                                                                     HisIleSerLysSerLysValGlyTrpLeuIleGlnLeuPheHisLys                              145150155160                                                                  LysIleGluSerAlaLeuArgAsnLysMetAsnSerGlnValCysGlu                              165170175                                                                     LysValThrAsnSerValSerSerLysLeuGlnProTyrPheGlnThr                              180185190                                                                     LeuProValMetThrLysIleAspSerValAlaGlyIleAsnTyrGly                              195200205                                                                     LeuValAlaProProAlaThrThrAlaGluThrLeuAspValGlnMet                              210215220                                                                     LysGlyGluPheTyrSerGluAsnHisHisAsnProProProPheAla                              225230235240                                                                  ProProValMetGluPheProAlaAlaHisAspArgMetValTyrLeu                              245250255                                                                     GlyLeuSerAspTyrPhePheAsnThrAlaGlyLeuValTyrGlnGlu                              260265270                                                                     AlaGlyValLeuLysMetThrLeuArgAspAspMetIleProLysGlu                              275280285                                                                     SerLysPheArgLeuThrThrLysPhePheGlyThrPheLeuProGlu                              290295300                                                                     ValAlaLysLysPheProAsnMetLysIleGlnIleHisValSerAla                              305310315320                                                                  SerThrProProHisLeuSerValGlnProThrGlyLeuThrPheTyr                              325330335                                                                     ProAlaValAspValGlnAlaPheAlaValLeuProAsnSerAlaLeu                              340345350                                                                     AlaSerLeuPheLeuIleGlyMetHisThrThrGlySerMetGluVal                              355360365                                                                     SerAlaGluSerAsnArgLeuValGlyGluLeuLysLeuAspArgLeu                              370375380                                                                     LeuLeuGluLeuLysHisSerAsnIleGlyProPheProValGluLeu                              385390395400                                                                  LeuGlnAspIleMetAsnTyrIleValProIleLeuValLeuProArg                              405410415                                                                     ValAsnGluLysLeuGlnLysGlyPheProLeuProThrProAlaArg                              420425430                                                                     ValGlnLeuTyrAsnValValLeuGlnProHisGlnAsnPheLeuLeu                              435440445                                                                     PheGlyAlaAspValValTyrLys                                                      450455                                                                        (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 456 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      ValAsnProGlyValValValArgIleSerGlnLysGlyLeuAspTyr                              151015                                                                        AlaSerGlnGlnGlyThrAlaAlaLeuGlnLysGluLeuLysArgIle                              202530                                                                        LysIleProAspTyrSerAspSerPheLysIleLysHisLeuGlyLys                              354045                                                                        GlyHisTyrSerPheTyrSerMetAspIleArgGluPheGlnLeuPro                              505560                                                                        SerSerGlnIleSerMetValProAsnValGlyLeuLysPheSerIle                              65707580                                                                      SerAsnAlaAsnIleLysIleSerGlyLysTrpLysAlaGlnLysArg                              859095                                                                        PheLeuLysMetSerGlyAsnPheAspLeuSerIleGluGlyMetSer                              100105110                                                                     IleSerAlaAspLeuLysLeuGlySerAsnProThrSerGlyLysPro                              115120125                                                                     ThrIleThrCysSerSerCysSerSerHisIleAsnSerValHisVal                              130135140                                                                     HisIleSerLysSerLysValGlyTrpLeuIleGlnLeuPheHisLys                              145150155160                                                                  LysIleGluSerAlaLeuArgAsnLysMetAsnSerGlnValCysGlu                              165170175                                                                     LysValThrAsnSerValSerSerLysLeuGlnProTyrPheGlnThr                              180185190                                                                     LeuProValMetThrLysIleAspProValAlaGlyIleAsnTyrGly                              195200205                                                                     LeuValAlaProProAlaThrThrAlaGluThrLeuAspValGlnMet                              210215220                                                                     LysGlyGluPheTyrSerGluAsnHisHisAsnProProProPheAla                              225230235240                                                                  ProProValMetGluPheProAlaAlaHisAspArgMetValTyrLeu                              245250255                                                                     GlyLeuSerAspTyrPhePheAsnThrAlaGlyLeuValTyrGlnGlu                              260265270                                                                     AlaGlyValLeuLysMetThrLeuArgAspAspMetIleProLysGlu                              275280285                                                                     SerLysPheArgLeuThrThrLysPhePheGlyThrPheLeuProGlu                              290295300                                                                     ValAlaLysLysPheProAsnMetLysIleGlnIleHisValSerAla                              305310315320                                                                  SerThrProProHisLeuSerValGlnProThrGlyLeuThrPheTyr                              325330335                                                                     ProAlaValAspValGlnAlaPheAlaValLeuProAsnSerSerLeu                              340345350                                                                     AlaSerLeuPheLeuIleGlyMetHisThrThrGlySerMetGluVal                              355360365                                                                     SerAlaGluSerAsnArgLeuValGlyGluLeuLysLeuAspArgLeu                              370375380                                                                     LeuLeuGluLeuLysHisSerAsnIleGlyProPheProValGluLeu                              385390395400                                                                  LeuGlnAspIleMetAsnTyrIleValProIleLeuValLeuProArg                              405410415                                                                     ValAsnGluLysLeuGlnLysGlyPheProLeuProThrProAlaArg                              420425430                                                                     ValGlnLeuTyrAsnValValLeuGlnProHisGlnAsnPheLeuLeu                              435440445                                                                     PheGlyAlaAspValValTyrLys                                                      450455                                                                        (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      TATCATGCTAG11                                                                 (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      CAGGCCTTGAGGTTTTGGCAG21                                                       (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CTGCCAGTAATGACCAAAATCGATCCTGTGGCTGGAATC39                                     (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GATTCTGTGGCTGGAATC18                                                          (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GTCTGCAACAGATATTTACTTGAGCTCATGCAG33                                           (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 103 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      MetArgGluAsnMetAlaArgGlyProCysAsnAlaProArgTrpVal                              151015                                                                        SerLeuMetValLeuValAsnLysMetAsnSerGlnValCysGluLys                              202530                                                                        ValThrAsnSerValSerSerLysLeuGlnProTyrPheGlnThrLeu                              354045                                                                        ProValMetThrLysIleAspSerValAlaGlyIleAsnTyrGlyLeu                              505560                                                                        ValAlaProProAlaThrThrAlaGluThrLeuAspValGlnMetLys                              65707580                                                                      GlyGluPheTyrSerGluLeuGlnProHisGlnAsnPheLeuLeuPhe                              859095                                                                        GlyAlaAspValValTyrLys                                                         100                                                                           (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 432 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      CAGGCCTTGAGGTTTTGGCAGCTCTGGAGGATGAGAGAGAACATGGCCAGGGGCCCTTGC60                AACGCGCCGAGATGGGTGTCCCTGATGGTGCTCGTCAACAAGATGAACAGCCAGGTCTGC120               GAGAAAGTGACCAATTCTGTATCCTCCAAGCTGCAACCTTATTTCCAGACTCTGCCAGTA180               ATGACCAAAATAGATTCTGTGGCTGGAATCAACTATGGTCTGGTGGCACCTCCAGCAACC240               ACGGCTGAGACCCTGGATGTACAGATGAAGGGGGAGTTTTACAGTGAGCTTCAGCCTCAC300               CAGAACTTCCTGCTGTTCGGTGCAGACGTTGTCTATAAATGAAGGCACCAGGGGTGCCGG360               GGGCTGTCAGCCGCACCTGTTCCTGATGGGCTGTGGGGCACCGGCTGCCTTTCCCCAGGG420               AATCCTCTCCAG432                                                               (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      CTGCCAGTAATGACCAAAATC21                                                       __________________________________________________________________________

What is claimed is:
 1. A biologically active recombinant variant of bactericidal/permeability increasing protein (BPI) as shown in FIG. 23 (SEQ ID NO:17).
 2. A biologically active recombinant variant according to claim 1, wherein the variant (1) specifically binds to endotoxin, (2) competes with BPI Protein for binding to endotoxin, and (3) inhibits endotoxin-induced lethality. 